Compositions Comprising Enzyme-Cleavable Ketone-Modified Opioid Prodrugs and Optional Inhibitors Thereof

ABSTRACT

A method of providing a patient with controlled release of ketone-containing opioid using a prodrug capable, upon enzymatic activation and intramolecular cyclization, of releasing the ketone-containing opioid is disclosed. The disclosure also provides such prodrug compounds and pharmaceutical compositions comprising such compounds. Such pharmaceutical compositions can optionally include an enzyme inhibitor that interacts with the enzyme(s) to mediate the enzymatically-controlled release of the ketone-containing opioid from the prodrug so as to modify enzymatic cleavage of the prodrug. Also included are methods to use such compounds and pharmaceutical compositions.

INTRODUCTION

Ketone-containing opioids are susceptible to misuse, abuse, or overdose.Use of and access to these drugs therefore needs to be controlled. Thecontrol of access to the drugs is expensive to administer and can resultin denial of treatment for patients that are not able to presentthemselves for dosing. For example, patients suffering from acute painmay be denied treatment with an opioid unless they have been admitted toa hospital. Furthermore, control of use is often ineffective, leading tosubstantial morbidity and deleterious social consequences.

SUMMARY

The embodiments include compositions comprising a ketone-modified opioidprodrug, wherein the ketone-modified opioid prodrug comprises aketone-containing opioid covalently bound to a promoiety comprising atrypsin-cleavable moiety, wherein cleavage of the trypsin-cleavablemoiety by trypsin mediates release of the ketone-containing opioid; anda trypsin inhibitor that interacts with the trypsin that mediatesenzymatically-controlled release of the ketone-containing opioid fromthe ketone-modified opioid prodrug following ingestion of thecomposition. Such cleavage can initiate, contribute to or effectketone-containing opioid release.

The embodiments include dose units comprising compositions comprising aketone-modified opioid prodrug and a trypsin inhibitor, where theketone-modified opioid prodrug and trypsin inhibitor are present in thedose unit in an amount effective to provide for a pre-selectedpharmacokinetic (PK) profile following ingestion. In furtherembodiments, the pre-selected PK profile comprises at least one PKparameter value that is less than the PK parameter value ofketone-containing opioid released following ingestion of an equivalentdosage of ketone-modified opioid prodrug in the absence of inhibitor. Infurther embodiments, the PK parameter value is selected from aketone-containing opioid Cmax value, a ketone-containing opioid exposurevalue, and a (1/ketone-containing opioid Tmax) value.

In certain embodiments, the dose unit provides for a pre-selected PKprofile following ingestion of at least two dose units. In relatedembodiments, the pre-selected PK profile of such dose units is modifiedrelative to the PK profile following ingestion of an equivalent dosageof ketone-modified opioid prodrug without inhibitor. In relatedembodiments, such a dose unit provides that ingestion of an increasingnumber of the dose units provides for a linear PK profile.

In related embodiments, such a dose unit provides that ingestion of anincreasing number of the dose units provides for a nonlinear PK profile.In related embodiments, the PK parameter value of the PK profile of sucha dose units is selected from a ketone-containing opioid Cmax value, a(1/ketone-containing opioid Tmax) value, and a ketone-containing opioidexposure value.

The embodiments include compositions comprising a container suitable forcontaining a composition for administration to a patient; and a doseunit as described herein disposed within the container.

The embodiments include dose units of a ketone-modified opioid prodrugand a trypsin inhibitor wherein the dose unit has a total weight of from1 microgram to 2 grams. The embodiments include pharmaceuticalcompositions of a ketone-modified opioid prodrug and a trypsin inhibitorwherein the combined weight of ketone-modified opioid prodrug andtrypsin inhibitor is from 0.1% to 99% per gram of the composition.

The embodiments include compositions and dose units wherein theketone-modified opioid prodrug is a compound of formula KC-(Ia):

wherein:

R^(a) is hydrogen or hydroxyl;

R⁵ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl, substituted cycloalkyl, aryl, or substituted aryl group, ortwo R¹ or R² groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, form a cycloalkyl, substitutedcycloalkyl, aryl, or substituted aryl group;

n is an integer from 2 to 4;

R³ is hydrogen;

R⁴ is

each R⁶ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring;

each W is independently —NR⁸—, —O— or —S—;

each R⁸ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl and substituted aryl, or optionally, each R⁶ and R⁸independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring;

p is an integer from one to 100; and

R⁷ is selected from hydrogen, alkyl, substituted alkyl, acyl,substituted acyl, alkoxycarbonyl, substituted alkoxycarbonyl, aryl,substituted aryl, arylalkyl, and substituted arylalkyl;

or a salt, hydrate or solvate thereof.

The embodiments include compositions and dose units wherein theketone-modified opioid prodrug is a compound of formula KC-(Ib):

wherein:

R^(a) is hydrogen or hydroxyl;

R⁵ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl or substituted cycloalkyl group, or two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n is an integer from 2 to 4;

R³ is hydrogen;

R⁴ is

each R⁶ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring;

each W is independently —NR⁸—, —O— or —S—;

each R⁸ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl and substituted aryl, or optionally, each R⁶ and R⁸independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring;

p is an integer from one to 100; and

R⁷ is selected from hydrogen, alkyl, substituted alkyl, acyl,substituted acyl, alkoxycarbonyl, substituted alkoxycarbonyl, aryl,substituted aryl, arylalkyl, and substituted arylalkyl;

or a salt, hydrate or solvate thereof.

The embodiments include compositions and dose units wherein theketone-modified opioid prodrug is a compound of formula KC-(II):

wherein:

R^(a) is hydrogen or hydroxyl;

R⁵ is selected from (1-6C)alkyl, (1-6C) substituted alkyl,—(CH₂)_(q)(C₆H₄)—COOH, —(CH₂)_(q)(C₆H₄)—COOCH₃, and—(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is an integer from one to 10;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl or substituted cycloalkyl group, or two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n is 2 or 3;

R³ is hydrogen;

R⁴ is a residue of an L-amino acid selected from alanine, arginine,asparagine, aspartic acid, cysteine, glycine, glutamine, glutamic acid,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine and valine, or aresidue of an N-acyl derivative of any of said amino acids; or a residueof a peptide composed of at least two L-amino acid residues selectedindependently from alanine, arginine, asparagine, aspartic acid,cysteine, glycine, glutamine, glutamic acid, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine or a residue of an N-acyl derivativethereof.

The embodiments include compositions and dose units wherein theketone-modified opioid prodrug is a compound of formula KC-(IIIa):

wherein:

X represents a residue of a ketone-containing opioid, wherein thehydrogen atom of the corresponding enolic group of the ketone isreplaced by a covalent bond to —C(O)—NR⁵—(C(R¹)(R²))_(n)—NR³R⁴;

R⁵ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl, substituted cycloalkyl, aryl, or substituted aryl group, ortwo R² or R³ groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, form a cycloalkyl, substitutedcycloalkyl, aryl, or substituted aryl group;

n is an integer from 2 to 4;

R³ is hydrogen;

R⁴ is

each R⁶ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring;

each W is independently —NR⁸—, —O— or —S—;

each R⁸ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl and substituted aryl, or optionally, each R⁶ and R⁸independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring;

p is an integer from one to 100; and

R⁷ is selected from hydrogen, alkyl, substituted alkyl, acyl,substituted acyl, alkoxycarbonyl, substituted alkoxycarbonyl, aryl,substituted aryl, arylalkyl, and substituted arylalkyl;

or a salt, hydrate or solvate thereof.

The embodiments include compositions and dose units wherein theketone-modified opioid prodrug is a compound of formula KC-(IIIb):

wherein:

X represents a residue of a ketone-containing opioid, wherein thehydrogen atom of the corresponding enolic group of the ketone isreplaced by a covalent bond to —C(O)—NR⁵—(C(R¹)(R²))_(n)—NR³R⁴;

R⁵ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl or substituted cycloalkyl group, or two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n is an integer from 2 to 4;

R³ is hydrogen;

R⁴ is

each R⁶ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring;

each W is independently —NR⁸—, —O— or —S—;

each R⁸ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl and substituted aryl, or optionally, each R⁶ and R⁸independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring;

p is an integer from one to 100; and

R⁷ is selected from hydrogen, alkyl, substituted alkyl, acyl,substituted acyl, alkoxycarbonyl, substituted alkoxycarbonyl, aryl,substituted aryl, arylalkyl, and substituted arylalkyl;

or a salt, hydrate or solvate thereof.

The embodiments include compositions and dose units wherein theketone-modified opioid prodrug is a compound of formula KC-(IV):

wherein:

X represents a residue of a ketone-containing opioid, wherein thehydrogen atom of the corresponding enolic group of the ketone isreplaced by a covalent bond to —C(O)—NR⁵—(C(R¹)(R²))_(n)—NR³R⁴;

R⁵ is selected from (1-6C)alkyl, (1-6C) substituted alkyl,—(CH₂)_(q)(C₆H₄)—COOH, —(CH₂)_(q)(C₆H₄)—COOCH₃, and—(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is an integer from one to 10;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl or substituted cycloalkyl group, or two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n is 2 or 3;

R³ is hydrogen;

R⁴ is a residue of an L-amino acid selected from alanine, arginine,asparagine, aspartic acid, cysteine, glycine, glutamine, glutamic acid,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine and valine, or aresidue of an N-acyl derivative of any of said amino acids; or a residueof a peptide composed of at least two L-amino acid residues selectedindependently from alanine, arginine, asparagine, aspartic acid,cysteine, glycine, glutamine, glutamic acid, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine or a residue of an N-acyl derivativethereof;

or a salt, hydrate or solvate thereof.

The embodiments include compositions and dose units wherein theketone-modified opioid prodrug is a compound of formula KC-(V):

wherein:

X represents a residue of a ketone-containing opioid, wherein thehydrogen atom of the corresponding enolic group of the ketone isreplaced by a covalent bond to —C(O)—NR⁵—(C(R¹)(R²))_(n)—NR³R⁴;

R⁵ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl, substituted cycloalkyl, aryl, or substituted aryl group, ortwo R¹ or R² groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, form a cycloalkyl, substitutedcycloalkyl, aryl, or substituted aryl group;

n is an integer from 2 to 4;

R³ is hydrogen;

R⁴ is a trypsin-cleavable moiety;

or a salt, hydrate or solvate thereof.

The embodiments include methods for treating a patient comprisingadministering any of the compositions or dose units described herein toa patient in need thereof. The embodiments include methods to reduceside effects of a therapy comprising administering any of thecompositions or dose units described herein to a patient in needthereof. The embodiments include methods of improving patient compliancewith a therapy prescribed by a clinician comprising directingadministration of any of the compositions or dose units described hereinto a patient in need thereof. Such embodiments can provide for improvedpatient compliance with a prescribed therapy as compared to patientcompliance with a prescribed therapy using drug and/or using prodrugwithout inhibitor as compared to prodrug with inhibitor.

The embodiments include methods of reducing risk of unintended overdoseof a ketone-containing opioid comprising directing administration of anyof the pharmaceutical compositions or dose units described herein to apatient in need of treatment.

The embodiments include methods of making a dose unit comprisingcombining a ketone-modified opioid prodrug and a trypsin inhibitor in adose unit, wherein the ketone-modified opioid prodrug and trypsininhibitor are present in the dose unit in an amount effective toattenuate release of the ketone-containing opioid from theketone-modified opioid prodrug.

The embodiments include methods of deterring misuse or abuse of multipledose units of a ketone-modified opioid prodrug comprising combining aketone-modified opioid prodrug and a trypsin inhibitor in a dose unit,wherein the ketone-modified opioid prodrug and trypsin inhibitor arepresent in the dose unit in an amount effective to attenuate release ofthe ketone-containing opioid from the ketone-modified opioid prodrugsuch that ingestion of multiples of dose units by a patient does notprovide a proportional release of ketone-containing opioid. In furtherembodiments, release of drug is decreased compared to release of drug byan equivalent dosage of prodrug in the absence of inhibitor.

One embodiment is a method for identifying a prodrug and a GI enzymeinhibitor suitable for formulation in a dose unit. Such a method can beconducted as, for example, an in vitro assay, an in vivo assay, or an exvivo assay.

The embodiments include methods for identifying a ketone-modified opioidprodrug and a trypsin inhibitor suitable for formulation in a dose unitcomprising combining a ketone-modified opioid prodrug, a trypsininhibitor, and trypsin in a reaction mixture, and detectingketone-modified opioid prodrug conversion, wherein a decrease inketone-modified opioid prodrug conversion in the presence of the trypsininhibitor as compared to ketone-modified opioid prodrug conversion inthe absence of the trypsin inhibitor indicates the ketone-modifiedopioid prodrug and trypsin inhibitor are suitable for formulation in adose unit.

The embodiments include methods for identifying a ketone-modified opioidprodrug and a trypsin inhibitor suitable for formulation in a dose unitcomprising administering to an animal a ketone-modified opioid prodrugand a trypsin inhibitor and detecting ketone-modified opioid prodrugconversion, wherein a decrease in ketone-containing opioid conversion inthe presence of the trypsin inhibitor as compared to ketone-containingopioid conversion in the absence of the trypsin inhibitor indicates theketone-modified opioid prodrug and trypsin inhibitor are suitable forformulation in a dose unit. In certain embodiments, administeringcomprises administering to the animal increasing doses of inhibitorco-dosed with a selected fixed dose of ketone-modified opioid prodrug.Detecting prodrug conversion can facilitate identification of a dose ofinhibitor and a dose of ketone-modified opioid prodrug that provides fora pre-selected pharmacokinetic (PK) profile. Such methods can beconducted as, for example, an in vivo assay or an ex vivo assay.

The embodiments include methods for identifying a ketone-modified opioidprodrug and a trypsin inhibitor suitable for formulation in a dose unitcomprising administering to an animal tissue a ketone-modified opioidprodrug and a trypsin inhibitor and detecting ketone-modified opioidprodrug conversion, wherein a decrease in ketone-modified opioid prodrugconversion in the presence of the trypsin inhibitor as compared toketone-modified opioid prodrug conversion in the absence of the trypsininhibitor indicates the ketone-modified opioid prodrug and trypsininhibitor are suitable for formulation in a dose unit.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representing the effect of increasing the level ofa GI enzyme inhibitor (“inhibitor”, X axis) on a PK parameter (e.g.,drug Cmax) (Y axis) for a fixed dose of prodrug. The effect of inhibitorupon a prodrug PK parameter can range from undetectable, to moderate, tocomplete inhibition (i.e., no detectable drug release).

FIG. 2 provides schematics of drug concentration in plasma (Y axis) overtime (X axis). Panel A is a schematic of a pharmacokinetic (PK) profilefollowing ingestion of prodrug with a GI enzyme inhibitor (dashed line)where the drug Cmax is modified relative to that of prodrug withoutinhibitor (solid line). Panel B is a schematic of a PK profile followingingestion of prodrug with inhibitor (dashed line) where drug Cmax anddrug Tmax are modified relative to that of prodrug without inhibitor(solid line). Panel C is a schematic of a PK profile following ingestionof prodrug with inhibitor (dashed line) where drug Tmax is modifiedrelative to that of prodrug without inhibitor (solid line).

FIG. 3 provides schematics representing differential concentration-dosePK profiles that can result from the dosing of multiples of a dose unit(X axis) of the present disclosure. Different PK profiles (asexemplified herein for a representative PK parameter, drug Cmax (Yaxis)) can be provided by adjusting the relative amount of prodrug andGI enzyme inhibitor contained in a single dose unit or by using adifferent prodrug or inhibitor in the dose unit.

FIG. 4 shows a plasma concentration time course of the production ofoxycodone following oral (PO) dosing of an oxycodone prodrug in rats.

FIG. 5 shows a plasma concentration time course of the production ofoxycodone following intravenous (IV) dosing of an oxycodone prodrug inrats.

FIG. 6 shows release of oxycodone from an oxycodone prodrug exposed to avariety of readily availably household chemicals or enzyme preparations.

FIG. 7 shows disappearance of an oxycodone prodrug and appearance ofoxycodone following in vitro incubation of the prodrug and trypsin, inthe absence or presence of a trypsin inhibitor.

FIG. 8 compares mean plasma concentrations over time of oxycodonerelease following PO administration of prodrug Compound KC-2 alone andCompound KC-2 with trypsin inhibitor Compound 109 to rats.

FIG. 9 compares mean plasma concentrations over time of oxycodonerelease following PO administration of increasing doses of prodrugCompound KC-2 to rats.

FIG. 10 compares mean plasma concentrations over time of oxycodonerelease following PO administration of prodrug Compound KC-2 withincreasing amounts of co-dosed trypsin inhibitor Compound 109 to rats.

FIG. 11 compares mean plasma concentrations over time of oxycodonerelease following PO administration of increasing doses of Compound KC-3to rats.

FIG. 12 shows a plasma concentration time course of the production ofoxycodone following intravenous (IV) dosing of prodrug Compound KC-3 inrats.

FIG. 13 compares mean plasma concentrations over time of oxycodonerelease following PO administration of prodrug Compound KC-3 withincreasing amounts of co-dosed trypsin inhibitor Compound 109 to rats.

FIG. 14 demonstrates the release of oxycodone from prodrug Compound KC-3exposed to a variety of household chemicals and enzyme preparations.

FIG. 15 shows a plasma concentration time course of the production ofoxycodone following intravenous (IV) dosing of prodrug Compound KC-4 inrats.

FIG. 16 compares mean plasma concentrations over time of hydrocodonerelease following PO administration of prodrug Compound KC-4 with orwithout a co-dose of trypsin inhibitor to rats.

FIG. 17 demonstrates mean plasma concentrations over time of oxycodonerelease following PO administration of Compound KC-5 to rats.

FIG. 18 shows a plasma concentration time course of the production ofoxycodone following intravenous (IV) dosing of prodrug Compound KC-5 inrats.

FIG. 19 demonstrates mean plasma concentrations over time of oxycodonerelease following PO administration of Compound KC-6 to rats.

FIG. 20 shows a plasma concentration time course of the production ofoxycodone following intravenous (IV) dosing of prodrug Compound KC-6 inrats.

DEFINITIONS

The following terms have the following meaning unless otherwiseindicated. Any undefined terms have their art recognized meanings.

As used herein, the term “alkyl” by itself or as part of anothersubstituent refers to a saturated branched or straight-chain monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane. Typical alkyl groups include, butare not limited to, methyl; ethyl, propyls such as propan-1-yl orpropan-2-yl; and butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl or 2-methyl-propan-2-yl. In some embodiments, analkyl group comprises from 1 to 20 carbon atoms. In other embodiments,an alkyl group comprises from 1 to 10 carbon atoms. In still otherembodiments, an alkyl group comprises from 1 to 6 carbon atoms, such asfrom 1 to 4 carbon atoms.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl radical derived bythe removal of one hydrogen atom from a single carbon atom of an alkane.Typical alkanyl groups include, but are not limited to, methanyl;ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl),cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, etc.; and the like.

“Alkylene” refers to a branched or unbranched saturated hydrocarbonchain, usually having from 1 to 40 carbon atoms, more usually 1 to 10carbon atoms and even more usually 1 to 6 carbon atoms. This term isexemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon double bond derived by the removal of onehydrogen atom from a single carbon atom of an alkene. The group may bein either the cis or trans conformation about the double bond(s).Typical alkenyl groups include, but are not limited to, ethenyl;propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl(allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl;butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl,buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl,cyclobuta-1,3-dien-1-yl, etc.; and the like.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl radical having atleast one carbon-carbon triple bond derived by the removal of onehydrogen atom from a single carbon atom of an alkyne. Typical alkynylgroups include, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

“Acyl” by itself or as part of another substituent refers to a radical—C(O)R³⁰, where R³⁰ is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl,aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as definedherein and substituted versions thereof. Representative examplesinclude, but are not limited to formyl, acetyl, cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, piperonyl, succinyl,and malonyl, and the like.

The term “aminoacyl” refers to the group —C(O)NR²¹R²², wherein R²¹ andR²² independently are selected from the group consisting of hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic andwhere R²¹ and R²² are optionally joined together with the nitrogen boundthereto to form a heterocyclic or substituted heterocyclic group, andwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Alkoxy” by itself or as part of another substituent refers to a radical—OR³¹ where R³¹ represents an alkyl or cycloalkyl group as definedherein. Representative examples include, but are not limited to,methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy and the like.

“Alkoxycarbonyl” by itself or as part of another substituent refers to aradical —C(O)OR³ where R³¹ represents an alkyl or cycloalkyl group asdefined herein. Representative examples include, but are not limited to,methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,cyclohexyloxycarbonyl and the like.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of an aromatic ring system.Typical aryl groups include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene and the like. In certain embodiments, an aryl groupcomprises from 6 to 20 carbon atoms. In certain embodiments, an arylgroup comprises from 6 to 12 carbon atoms. Examples of an aryl group arephenyl and naphthyl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylalkenyl and/orarylalkynyl is used. In certain embodiments, an arylalkyl group is(C₇-C₃₀) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C₁-C₁₀) and the aryl moiety is (C₆-C₂₀). In certainembodiments, an arylalkyl group is (C₇-C₂₀) arylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₈) andthe aryl moiety is (C₆-C₁₂).

“Arylaryl” by itself or as part of another substituent, refers to amonovalent hydrocarbon group derived by the removal of one hydrogen atomfrom a single carbon atom of a ring system in which two or moreidentical or non-identical aromatic ring systems are joined directlytogether by a single bond, where the number of such direct ringjunctions is one less than the number of aromatic ring systems involved.Typical arylaryl groups include, but are not limited to, biphenyl,triphenyl, phenyl-napthyl, binaphthyl, biphenyl-napthyl, and the like.When the number of carbon atoms in an arylaryl group are specified, thenumbers refer to the carbon atoms comprising each aromatic ring. Forexample, (C₅-C₁₄) arylaryl is an arylaryl group in which each aromaticring comprises from 5 to 14 carbons, e.g., biphenyl, triphenyl,binaphthyl, phenylnapthyl, etc. In certain embodiments, each aromaticring system of an arylaryl group is independently a (C₅-C₁₄) aromatic.In certain embodiments, each aromatic ring system of an arylaryl groupis independently a (C₅-C₁₀) aromatic. In certain embodiments, eacharomatic ring system is identical, e.g., biphenyl, triphenyl,binaphthyl, trinaphthyl, etc.

“Cycloalkyl” by itself or as part of another substituent refers to asaturated or unsaturated cyclic alkyl radical. Where a specific level ofsaturation is intended, the nomenclature “cycloalkanyl” or“cycloalkenyl” is used. Typical cycloalkyl groups include, but are notlimited to, groups derived from cyclopropane, cyclobutane, cyclopentane,cyclohexane and the like. In certain embodiments, the cycloalkyl groupis (C₃-C₁₀) cycloalkyl. In certain embodiments, the cycloalkyl group is(C₃-C₇) cycloalkyl.

“Cycloheteroalkyl” or “heterocyclyl” by itself or as part of anothersubstituent, refers to a saturated or unsaturated cyclic alkyl radicalin which one or more carbon atoms (and any associated hydrogen atoms)are independently replaced with the same or different heteroatom.Typical heteroatoms to replace the carbon atom(s) include, but are notlimited to, N, P, O, S, Si, etc. Where a specific level of saturation isintended, the nomenclature “cycloheteroalkanyl” or “cycloheteroalkenyl”is used. Typical cycloheteroalkyl groups include, but are not limitedto, groups derived from epoxides, azirines, thiiranes, imidazolidine,morpholine, piperazine, piperidine, pyrazolidine, pyrrolidine,quinuclidine and the like.

“Heteroalkyl, Heteroalkanyl, Heteroalkenyl and Heteroalkynyl” bythemselves or as part of another substituent refer to alkyl, alkanyl,alkenyl and alkynyl groups, respectively, in which one or more of thecarbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatomic groups. Typicalheteroatomic groups which can be included in these groups include, butare not limited to, —O—, —S—, —S—S—, —O—S—, —NR³⁷R³⁸—, .═N—N═, —N═N—,—N═N—NR³⁹R⁴⁰, —PR⁴¹—, —P(O)₂—, —POR⁴²—, —O—P(O)₂—, —S—O—, —S—(O)—,—SO₂—, —SnR⁴³R⁴⁴— and the like, where R³⁷, R³⁸, R³⁹, R⁴⁰, R⁴¹, R⁴², R⁴³and R⁴⁴ are independently hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl,substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl or substituted heteroarylalkyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a heteroaromatic ring system. Typicalheteroaryl groups include, but are not limited to, groups derived fromacridine, arsindole, carbazole, 3-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene,benzodioxole and the like. In certain embodiments, the heteroaryl groupis from 5-20 membered heteroaryl. In certain embodiments, the heteroarylgroup is from 5-10 membered heteroaryl. In certain embodiments,heteroaryl groups are those derived from thiophene, pyrrole,benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole,oxazole and pyrazine.

“Heteroarylalkyl” by itself or as part of another substituent, refers toan acyclic alkyl radical in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylalkenyl and/orheterorylalkynyl is used. In certain embodiments, the heteroarylalkylgroup is a 6-30 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is 1-10 membered and theheteroaryl moiety is a 5-20-membered heteroaryl. In certain embodiments,the heteroarylalkyl group is 6-20 membered heteroarylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is 1-8membered and the heteroaryl moiety is a 5-12-membered heteroaryl.

“Aromatic Ring System” by itself or as part of another substituent,refers to an unsaturated cyclic or polycyclic ring system having aconjugated π electron system. Specifically included within thedefinition of “aromatic ring system” are fused ring systems in which oneor more of the rings are aromatic and one or more of the rings aresaturated or unsaturated, such as, for example, fluorene, indane,indene, phenalene, etc. Typical aromatic ring systems include, but arenot limited to, aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, coronene, fluoranthene,fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene,indane, indene, naphthalene, octacene, octaphene, octalene, ovalene,penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene,phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene,triphenylene, trinaphthalene and the like.

“Heteroaromatic Ring System” by itself or as part of anothersubstituent, refers to an aromatic ring system in which one or morecarbon atoms (and any associated hydrogen atoms) are independentlyreplaced with the same or different heteroatom. Typical heteroatoms toreplace the carbon atoms include, but are not limited to, N, P, O, S,Si, etc. Specifically included within the definition of “heteroaromaticring systems” are fused ring systems in which one or more of the ringsare aromatic and one or more of the rings are saturated or unsaturated,such as, for example, arsindole, benzodioxan, benzofuran, chromane,chromene, indole, indoline, xanthene, etc. Typical heteroaromatic ringsystems include, but are not limited to, arsindole, carbazole,3-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene and the like.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Typical substituents include, but are not limited to, alkylenedioxy(such as methylenedioxy), -M, —R⁶⁰, —O⁻, ═O, —OR⁶⁰, —SR⁶⁰, —S—, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R⁶⁰, —OS(O)₂O, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹,—C(O)O—, —C(S)OR⁶⁰, —NR⁶²C(O)NR⁶⁰R⁶¹, —NR⁶²C(S)NR⁶⁰R⁶¹,—NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and —C(NR⁶²)NR⁶⁰R⁶¹ where M is halogen; R⁶⁰, R⁶¹,R⁶² and R⁶³ are independently hydrogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independentlyhydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, heteroaryl or substituted heteroaryl, or optionallyR⁶⁴ and R⁶⁵ together with the nitrogen atom to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certainembodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰, —S—, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂R⁶⁰,—OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹),—C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —NR⁶²C(O)NR⁶⁰R⁶¹.In certain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —P(O)(OR⁶⁰)(O⁻),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻. Incertain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰,—C(O)OR⁶⁰, —C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as defined above. Forexample, a substituted group may bear a methylenedioxy substituent orone, two, or three substituents selected from a halogen atom, a(1-4C)alkyl group and a (1-4C)alkoxy group.

“Dose unit” as used herein refers to a combination of a GIenzyme-cleavable prodrug (e.g., trypsin-cleavable prodrug) and a GIenzyme inhibitor (e.g., a trypsin inhibitor). A “single dose unit” is asingle unit of a combination of a GI enzyme-cleavable prodrug (e.g.,trypsin-cleavable prodrug) and a GI enzyme inhibitor (e.g., trypsininhibitor), where the single dose unit provide a therapeuticallyeffective amount of drug (i.e., a sufficient amount of drug to effect atherapeutic effect, e.g., a dose within the respective drug'stherapeutic window, or therapeutic range). “Multiple dose units” or“multiples of a dose unit” or a “multiple of a dose unit” refers to atleast two single dose units.

“PK profile” refers to a profile of drug concentration in blood orplasma. Such a profile can be a relationship of drug concentration overtime (i.e., a “concentration-time PK profile”) or a relationship of drugconcentration versus number of doses ingested (i.e., a“concentration-dose PK profile”). A PK profile is characterized by PKparameters.

“PK parameter” refers to a measure of drug concentration in blood orplasma, such as: 1) “drug Cmax”, the maximum concentration of drugachieved in blood or plasma; 2) “drug Tmax”, the time elapsed followingingestion to achieve Cmax; and 3) “drug exposure”, the totalconcentration of drug present in blood or plasma over a selected periodof time, which can be measured using the area under the curve (AUC) of atime course of drug release over a selected period of time (t).Modification of one or more PK parameters provides for a modified PKprofile.

“Pharmacodynamic (PD) profile” refers to a profile of the efficacy of adrug in a patient (or subject or user), which is characterized by PDparameters. “PD parameters” include “drug Emax” (the maximum drugefficacy), “drug EC50” (the concentration of drug at 50% of the Emax)and side effects.

“Gastrointestinal enzyme” or “GI enzyme” refers to an enzyme located inthe gastrointestinal (GI) tract, which encompasses the anatomical sitesfrom mouth to anus. Trypsin is an example of a GI enzyme.

“Gastrointestinal enzyme-cleavable moiety” or “GI enzyme-cleavablemoiety” refers to a group comprising a site susceptible to cleavage by aGI enzyme. For example, a “trypsin-cleavable moiety” refers to a groupcomprising a site susceptible to cleavage by trypsin.

“Gastrointestinal enzyme inhibitor” or “GI enzyme inhibitor” refers toany agent capable of inhibiting the action of a gastrointestinal enzymeon a substrate. The term also encompasses salts of gastrointestinalenzyme inhibitors. For example, a “trypsin inhibitor” refers to anyagent capable of inhibiting the action of trypsin on a substrate.

“Pharmaceutical composition” refers to at least one compound and canfurther comprise a pharmaceutically acceptable carrier, with which thecompound is administered to a patient.

“Pharmaceutically acceptable salt” refers to a salt of a compound, whichpossesses the desired pharmacological activity of the compound. Suchsalts include: (1) acid addition salts, formed with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the compound is replacedby a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, N-methylglucamine and the like.

The term “solvate” as used herein refers to a complex or aggregateformed by one or more molecules of a solute, e.g. a prodrug or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include by way of example, water, methanol, ethanol,isopropanol, acetic acid, and the like. When the solvent is water, thesolvate formed is a hydrate.

“Pharmaceutically acceptable carrier” refers to a diluent, adjuvant,excipient or vehicle with, or in which a compound is administered.

“Preventing” or “prevention” or “prophylaxis” refers to a reduction inrisk of occurrence of a condition, such as pain.

“Prodrug” refers to a derivative of an active agent that requires atransformation within the body to release the active agent. In certainembodiments, the transformation is an enzymatic transformation. Prodrugsare frequently, although not necessarily, pharmacologically inactiveuntil converted to the active agent.

“Promoiety” refers to a form of protecting group that when used to maska functional group within an active agent converts the active agent intoa prodrug. Typically, the promoiety will be attached to the drug viabond(s) that are cleaved by enzymatic or non-enzymatic means in vivo.

“Treating” or “treatment” of any condition, such as pain, refers, incertain embodiments, to ameliorating the condition (i.e., arresting orreducing the development of the condition). In certain embodiments“treating” or “treatment” refers to ameliorating at least one physicalparameter, which may not be discernible by the patient. In certainembodiments, “treating” or “treatment” refers to inhibiting thecondition, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In certain embodiments, “treating” or “treatment”refers to delaying the onset of the condition.

“Therapeutically effective amount” means the amount of a compound (e.g.,prodrug) that, when administered to a patient for preventing or treatinga condition such as pain, is sufficient to effect such treatment. The“therapeutically effective amount” will vary depending on the compound,the condition and its severity and the age, weight, etc., of thepatient.

DETAILED DESCRIPTION

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It should be understood that as used herein, the term “a” entity or “an”entity refers to one or more of that entity. For example, a compoundrefers to one or more compounds. As such, the terms “a”, “an”, “one ormore” and “at least one” can be used interchangeably. Similarly theterms “comprising”, “including” and “having” can be usedinterchangeably.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001.

The nomenclature used herein to name the subject compounds isillustrated in the Examples herein. When possible, this nomenclature hasgenerally been derived using the commercially-available AutoNom software(MDL, San Leandro, Calif.).

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterised, and tested for biological activity). In addition, allsub-combinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentinvention and are disclosed herein just as if each and every suchsub-combination of chemical groups was individually and explicitlydisclosed herein.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any of the means knownin the art, including chromatographic means, such as high performanceliquid chromatography (HPLC), preparative thin layer chromatography,flash column chromatography and ion exchange chromatography. Anysuitable stationary phase can be used, including normal and reversedphases as well as ionic resins. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, New York, 1969.

During any of the processes for preparation of the compounds of thepresent disclosure, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This canbe achieved by means of conventional protecting groups as described instandard works, such as T. W. Greene and P. G. M. Wuts, “ProtectiveGroups in Organic Synthesis”, Fourth edition, Wiley, New York 2006. Theprotecting groups can be removed at a convenient subsequent stage usingmethods known from the art.

The compounds described herein can contain one or more chiral centersand/or double bonds and therefore, can exist as stereoisomers, such asdouble-bond isomers (i.e., geometric isomers), enantiomers ordiastereomers. Accordingly, all possible enantiomers and stereoisomersof the compounds including the stereoisomerically pure form (e.g.,geometrically pure, enantiomerically pure or diastereomerically pure)and enantiomeric and stereoisomeric mixtures are included in thedescription of the compounds herein. Enantiomeric and stereoisomericmixtures can be resolved into their component enantiomers orstereoisomers using separation techniques or chiral synthesis techniqueswell known to the skilled artisan. The compounds can also exist inseveral tautomeric forms including the enol form, the keto form andmixtures thereof. Accordingly, the chemical structures depicted hereinencompass all possible tautomeric forms of the illustrated compounds.The compounds described also include isotopically labeled compoundswhere one or more atoms have an atomic mass different from the atomicmass conventionally found in nature. Examples of isotopes that can beincorporated into the compounds disclosed herein include, but are notlimited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, etc. Compounds canexist in unsolvated forms as well as solvated forms, including hydratedforms. In general, compounds can be hydrated or solvated. Certaincompounds can exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are equivalent for the uses contemplatedherein and are intended to be within the scope of the presentdisclosure.

Representative Embodiments

Reference will now be made in detail to various embodiments. It will beunderstood that the invention is not limited to these embodiments. Tothe contrary, it is intended to cover alternatives, modifications, andequivalents as may be included within the spirit and scope of theallowed claims.

The present disclosure provides pharmaceutical compositions, and theirmethods of use, where the pharmaceutical compositions comprise aketone-modified opioid prodrug that provides enzymatically-controlledrelease of a ketone-containing opioid and an optional enzyme inhibitorthat interacts with the enzyme(s) that mediates theenzymatically-controlled release of the ketone-containing opioid fromthe prodrug so as to attenuate enzymatic cleavage of the prodrug.

The disclosure provides pharmaceutical compositions which comprise anoptional trypsin inhibitor and a ketone-modified opioid prodrug thatcontains a trypsin-cleavable moiety that, when cleaved, facilitatesrelease of ketone-containing opioid.

According to one aspect, the embodiments include pharmaceuticalcompositions, which comprise a trypsin-cleavable ketone-modified opioidprodrug and an optional trypsin inhibitor.

Examples of ketone-modified opioid prodrugs and trypsin inhibitors aredescribed below.

Ketone-Containing Opioids

An “opioid” refers to a chemical substance that exerts itspharmacological action by interaction at an opioid receptor. An opioidcan be an isolated natural product, a synthetic compound or asemi-synthetic compound. “Ketone-containing opioid” refers to a subsetof the opioids that contain a ketone group. As used herein, aketone-containing opioid is an opioid containing an enolizable ketonegroup. A ketone-containing opioid is a compound with a pharmacophorethat presents to the opioid receptor an aromatic group and an aliphaticamine group in an architecturally discrete way. See, for example, Foye'sPrinciples of Medicinal Chemistry, Sixth Edition, ed. T. L. Lemke and D.A. Williams, Lippincott Williams & Wilkins, 2008, particularly Chapter24, pages 653-678.

For example, ketone-containing opioids include, but are not limited to,acetylmorphone, hydrocodone, hydromorphone, ketobemidone, methadone,naloxone, N-methylnaloxone, naltrexone, N-methylnaltrexone, oxycodone,oxymorphone, and pentamorphone.

In certain embodiments, the ketone-containing opioid is hydrocodone oroxycodone.

It is contemplated that opioids bearing at least some of thefunctionalities described herein will be developed; such opioids areincluded as part of the scope of this disclosure.

Ketone-Modified Opioid Prodrugs

The disclosure provides a ketone-modified opioid prodrug which providesenzymatically-controlled release of a ketone-containing opioid. In aketone-modified opioid prodrug, a promoiety is attached to theketone-containing opioid through the enolic oxygen atom of the ketonemoiety. In a ketone-modified opioid prodrug, the hydrogen atom of thecorresponding enolic group of the ketone-containing opioid is replacedby a covalent bond to a promoiety.

As disclosed herein, a trypsin-cleavable ketone-modified opioid prodrugis a ketone-modified opioid prodrug that comprises a promoietycomprising a trypsin-cleavable moiety, i.e., a moiety having a sitesusceptible to cleavage by trypsin. Such a prodrug comprises aketone-containing opioid covalently bound to a promoiety comprising atrypsin-cleavable moiety, wherein cleavage of the trypsin-cleavablemoiety by trypsin mediates release of the drug. Cleavage can initiate,contribute to or effect drug release.

Ketone-Modified Opioid Prodrugs with Promoiety Comprising CyclizableSpacer Leaving Group and Cleavable Moiety

According to certain embodiments, there is provided a ketone-modifiedopioid prodrug which provides enzymatically-controlled release of aketone-containing opioid. The disclosure provides for a ketone-modifiedopioid in which the promoiety comprises a cyclizable spacer leavinggroup and a cleavable moiety. In certain embodiments, theketone-containing opioid is a corresponding compound in which the enolicoxygen atom has a substituent which is a spacer leaving group bearing anitrogen nucleophile that is protected with an enzymatically-cleavablemoiety, the configuration of the spacer leaving group and nitrogennucleophile being such that, upon enzymatic cleavage of the cleavablemoiety, the nitrogen nucleophile is capable of forming a cyclic urea,liberating the compound from the spacer leaving group so as to provide aketone-containing opioid.

The corresponding prodrug provides post administration-activated,controlled release of the ketone-containing opioid. The prodrug requiresenzymatic cleavage to initiate release of the ketone-containing opioidand thus the rate of release of the ketone-containing opioid dependsupon both the rate of enzymatic cleavage and the rate of cyclization.Accordingly, the prodrug has reduced susceptibility to accidentaloverdosing or abuse, whether by deliberate overdosing, administrationthrough an inappropriate route, such as by injection, or by chemicalmodification using readily available household chemicals. The prodrug isconfigured so that it will not provide excessively high plasma levels ofthe active drug if it is administered inappropriately, and cannotreadily be decomposed to afford the active drug other than by enzymaticcleavage followed by controlled cyclization.

The enzyme-cleavable moiety linked to the nitrogen nucleophile throughan amide bond can be, for example, a residue of an amino acid or apeptide, or an (alpha)N-acyl derivative of an amino acid or peptide (forexample an N-acyl derivative of a pharmaceutically acceptable carboxylicacid). The peptide can contain, for example, up to about 100 amino acidresidues.

Each amino acid can advantageously be a naturally occurring amino acid,such as an L-amino acid. Examples of naturally occurring amino acids arealanine, arginine, asparagine, aspartic acid, cysteine, glycine,glutamine, glutamic acid, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine and valine. Accordingly, examples of enzyme-cleavable moietiesinclude residues of the L-amino acids listed hereinabove and N-acylderivatives thereof, and peptides formed from at least two of theL-amino acids listed hereinabove, and the N-acyl derivatives thereof.

The cyclic group formed when the ketone-containing opioid is released isconveniently pharmaceutically acceptable, in particular apharmaceutically acceptable cyclic urea. It will be appreciated thatcyclic ureas are generally very stable and have low toxicity.

Formulae KC-(I) and KC-(II)

The compositions of the present disclosure include compounds of formulaeKC-(I) and KC-(II) shown below. Compounds of formulae KC-(I) and KC-(II)are prodrugs of oxycodone and hydrocodone. Pharmaceutical compositionsand methods of the present disclosure also contemplate compounds offormulae KC-(I) and KC-(II).

Formula KC-(I)

In one of its composition aspects, the present embodiments provide acompound of formula KC-(Ia):

wherein:

R^(a) is hydrogen or hydroxyl;

R⁵ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl, substituted cycloalkyl, aryl, or substituted aryl group, ortwo R¹ or R² groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, form a cycloalkyl, substitutedcycloalkyl, aryl, or substituted aryl group;

n is an integer from 2 to 4;

R³ is hydrogen;

R⁴ is

each R⁶ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring;

each W is independently —NR⁸—, —O— or —S—;

each R⁸ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl and substituted aryl, or optionally, each R⁶ and R⁸independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring;

p is an integer from one to 100; and

R⁷ is selected from hydrogen, alkyl, substituted alkyl, acyl,substituted acyl, alkoxycarbonyl, substituted alkoxycarbonyl, aryl,substituted aryl, arylalkyl, and substituted arylalkyl;

or a salt, hydrate or solvate thereof.

In one of its composition aspects, the present embodiments provide acompound of formula KC-(Ib):

wherein:

R^(a) is hydrogen or hydroxyl;

R⁵ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl or substituted cycloalkyl group, or two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n is an integer from 2 to 4;

R³ is hydrogen;

R⁴ is

each R⁶ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring;

each W is independently —NR⁸—, —O— or —S—;

each R⁸ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl and substituted aryl, or optionally, each R⁶ and R⁸independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring;

p is an integer from one to 100; and

R⁷ is selected from hydrogen, alkyl, substituted alkyl, acyl,substituted acyl, alkoxycarbonyl, substituted alkoxycarbonyl, aryl,substituted aryl, arylalkyl, and substituted arylalkyl;

or a salt, hydrate or solvate thereof.

Reference to formula KC-(I) is meant to include compounds of formulaKC-(Ia) and KC-(Ib).

In formula KC-(I), R^(a) can be hydrogen or hydroxyl. In certaininstances, R^(a) is hydrogen. In other instances, R^(a) is hydroxyl.

In formula KC-(I), R⁵ can be selected from alkyl, substituted alkyl,arylalkyl, substituted arylalkyl, aryl and substituted aryl. In certaininstances, R⁵ is (1-6C)alkyl. In other instances, R⁵ is (1-4C)alkyl. Inother instances, R⁵ is methyl or ethyl. In other instances, R⁵ ismethyl. In certain instances, R⁵ is ethyl.

In certain instances, R⁵ is substituted alkyl. In certain instances, R⁵is an alkyl group substituted with a carboxylic group such as acarboxylic acid, carboxylic ester or carboxylic amide. In certaininstances, R⁵ is —(CH₂)_(n)—COOH, —(CH₂)_(n)—COOCH₃, or—(CH₂)_(n)—COOCH₂CH₃, wherein n is a number form one to 10. In certaininstances, R¹ is —(CH₂)₅—COOH, —(CH₂)₅—COOCH₃, or —(CH₂)₅—COOCH₂CH₃.

In certain instances, in formula KC-(I), R⁵ is arylalkyl or substitutedarylalkyl. In certain instances, in formula KC-(I), R⁵ is arylalkyl. Incertain instances, R⁵ is substituted arylalkyl. In certain instances, R⁵is an arylalkyl group substituted with a carboxylic group such as acarboxylic acid, carboxylic ester or carboxylic amide. In certaininstances, R⁵ is —(CH₂)_(q)(C₆H₄)—COOH, —(CH₂)_(q)(C₆H₄)—COOCH₃, or—(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is an integer from one to 10. Incertain instances, R⁵ is —CH₂(C₆H₄)—COOH, —CH₂(C₆H₄)—COOCH₃, or —CH₂(C₆H₄)—COOCH₂CH₃.

In certain instances, in formula KC-(I), R⁵ is aryl. In certaininstances, R⁵ is substituted aryl. In certain instances, R⁵ is an arylgroup ortho, meta or para-substituted with a carboxylic group such as acarboxylic acid, carboxylic ester or carboxylic amide. In certaininstances, R⁵ is —(C₆H₄)—COOH, —(C₆H₄)—COOCH₃, or —(C₆H₄)—COOCH₂CH₃.

In formula KC-(I), each R¹ can be independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, acyl, and aminoacyl.In certain instances, R¹ is hydrogen or alkyl. In certain instances, R¹is hydrogen. In certain instances, R¹ is alkyl. In certain instances, R¹is acyl. In certain instances, R¹ is aminoacyl.

In formula KC-(I), each R² can be independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, acyl, and aminoacyl.In certain instances, R² is hydrogen or alkyl. In certain instances, R²is hydrogen. In certain instances, R² is alkyl. In certain instances, R²is acyl. In certain instances, R² is aminoacyl.

In certain instances, R¹ and R² are hydrogen. In certain instances, R¹and R² on the same carbon are both alkyl. In certain instances, R¹ andR² on the same carbon are methyl. In certain instances, R¹ and R² on thesame carbon are ethyl.

In certain instances, R¹ and R¹ which are vicinal are both alkyl and R²and R² which are vicinal are both hydrogen. In certain instances, R¹ andR¹ which are vicinal are both ethyl and R² and R² which are vicinal areboth hydrogen. In certain instances, R¹ and R¹ which are vicinal areboth methyl and R² and R² which are vicinal are both hydrogen.

In certain instances, in the chain of —[C(R¹)(R²)]_(n)— in FormulaKC-(I), not every carbon is substituted. In certain instances, in thechain of —[C(R¹)(R²)]_(n)—, there is a combination of different alkylsubstituents, such as methyl or ethyl.

In certain instances, one of R¹ and R² is methyl, ethyl or other alkyland R⁵ is alkyl. In certain instances, R¹ and R¹ which are vicinal areboth alkyl and R² and R² which are vicinal are both hydrogen and R⁵ isalkyl. In certain instances, R¹ and R¹ which are vicinal are both ethyland R² and R² which are vicinal are both hydrogen and R⁵ is alkyl. Incertain instances, R¹ and R¹ which are vicinal are both methyl and R²and R² which are vicinal are both hydrogen and R⁵ is alkyl.

In certain instances, one of R¹ and R² is methyl, ethyl or other alkyland R⁵ is substituted alkyl. In certain instances, one of R¹ and R² ismethyl, ethyl or other alkyl and R⁵ is an alkyl group substituted with acarboxylic group such as a carboxylic acid, carboxylic ester orcarboxylic amide. In certain instances, one of R¹ and R² is methyl,ethyl or other alkyl and R⁵ is —(CH₂)_(q)(C₆H₄)—COOH,—(CH₂)_(q)(C₆H₄)—COOCH₃, or —(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is aninteger from one to 10. In certain instances, one of R¹ and R² ismethyl, ethyl or other alkyl and R⁵ is an alkyl group substituted withcarboxamide.

In formula KC-(I), R¹ and R² together with the carbon to which they areattached can form a cycloalkyl or substituted cycloalkyl group, or twoR¹ or R² groups on adjacent carbon atoms, together with the carbon atomsto which they are attached, can form a cycloalkyl or substitutedcycloalkyl group. In certain instances, R¹ and R² together with thecarbon to which they are attached can form a cycloalkyl group. Thus, incertain instances, R¹ and R² on the same carbon form a spirocycle. Incertain instances, R¹ and R² together with the carbon to which they areattached can form a substituted cycloalkyl group. In certain instances,two R¹ or R² groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, can form a cycloalkyl group. Incertain instances, two R¹ or R² groups on adjacent carbon atoms,together with the carbon atoms to which they are attached, can form asubstituted cycloalkyl group.

In formula KC-(I), R¹ and R² together with the carbon to which they areattached can form an aryl or substituted aryl group, or two R¹ or R²groups on adjacent carbon atoms, together with the carbon atoms to whichthey are attached, can form an aryl or substituted aryl group. Incertain instances, two R¹ or R² groups on adjacent carbon atoms,together with the carbon atoms to which they are attached, form a phenylring. In certain instances, two R¹ or R² groups on adjacent carbonatoms, together with the carbon atoms to which they are attached, form asubstituted phenyl ring. In certain instances, two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a naphthyl ring.

In certain instances, one of R¹ and R² is aminoacyl.

In certain instances, one or both of R¹ and R² is aminoacyl comprisingphenylenediamine.

In certain instances, one of R¹ and R² is

wherein each R¹ is independently selected from hydrogen, alkyl,substituted alkyl, and acyl and R¹¹ is alkyl or substituted alkyl. Incertain instances, at least one of R¹⁰ is acyl. In certain instances, atleast one of R¹⁰ is alkyl or substituted alkyl. In certain instances, atleast one of R¹⁰ is hydrogen. In certain instances, both of R¹⁰ arehydrogen.

In certain instances, one of R¹ and R² is

wherein R¹⁰ is hydrogen, alkyl, substituted alkyl, or acyl. In certaininstances, R¹⁰ is acyl. In certain instances, R¹⁰ is alkyl orsubstituted alkyl. In certain instances, R¹⁰ is hydrogen.

In certain instances, one of R¹ and R² is

wherein each R¹⁰ is independently hydrogen, alkyl, substituted alkyl, oracyl and b is a number from one to 5. In certain instances, one of R¹and R² is

wherein each R¹⁰ is independently hydrogen, alkyl, substituted alkyl, oracyl. In certain instances, one of R¹ and R² is

wherein R^(10a) is alkyl and each R¹⁰ is independently hydrogen, alkyl,substituted alkyl, or acyl.

In certain instances, one of R¹ and R² is

wherein R¹⁰ is independently hydrogen, alkyl, substituted alkyl, or acyland b is a number from one to 5. In certain instances, one of R¹ and R²is

wherein R¹⁰ is independently hydrogen, alkyl, substituted alkyl, oracyl.

In certain instances, one of R¹ and R² is an aminoacyl group, such as—C(O)NR^(10a)R^(10b) wherein each R^(10a) and R^(10b) is independentlyselected from hydrogen, alkyl, substituted alkyl, and acyl. In certaininstances, one of R¹ and R² is an aminoacyl group, such as—C(O)NR^(10a)R^(10b) wherein R^(10a) is an alkyl and R^(10b) issubstituted alkyl. In certain instances, one of R¹ and R² is anaminoacyl group, such as —C(O)NR^(10a)R^(10b), wherein R^(10a) is analkyl and R^(10b) is alkyl substituted with a carboxylic acid orcarboxyl ester. In certain instances, one of R¹ and R² is an aminoacylgroup, such as —C(O)NR^(10a)R^(10b), wherein R^(10a) is methyl andR^(10b) is alkyl substituted with a carboxylic acid or carboxyl ester.

In certain instances, R¹ or R² can modulate a rate of intramolecularcyclization. R¹ or R² can speed up a rate of intramolecular cyclization,when compared to the corresponding molecule where R¹ and R² are bothhydrogen. In certain instances, R¹ or R² comprise anelectron-withdrawing group or an electron-donating group. In certaininstances, R¹ or R² comprise an electron-withdrawing group. In certaininstances, R¹ or R² comprise an electron-donating group.

Atoms and groups capable of functioning as electron withdrawingsubstituents are well known in the field of organic chemistry. Theyinclude electronegative atoms and groups containing electronegativeatoms. Such groups function to lower the basicity or protonation stateof a nucleophilic nitrogen in the beta position via inductive withdrawalof electron density. Such groups can also be positioned on otherpositions along the alkylene chain. Examples include halogen atoms (forexample, a fluorine atom), acyl groups (for example an alkanoyl group,an aroyl group, a carboxyl group, an alkoxycarbonyl group, anaryloxycarbonyl group or an aminocarbonyl group (such as a carbamoyl,alkylaminocarbonyl, dialkylaminocarbonyl or arylaminocarbonyl group)),an oxo (═O) substituent, a nitrile group, a nitro group, ether groups(for example an alkoxy group) and phenyl groups bearing a substituent atthe ortho position, the para position or both the ortho and the parapositions, each substituent being selected independently from a halogenatom, a fluoroalkyl group (such as trifluoromethyl), a nitro group, acyano group and a carboxyl group. Each of the electron withdrawingsubstituents can be selected independently from these.

In certain instances, —[C(R¹)(R²)]_(n)— is selected from—CH(CH₂F)CH(CH₂F)—; —CH(CHF₂)CH(CHF₂)—; —CH(CF₃)CH(CF₃)—; —CH₂CH(CF₃)—;—CH₂CH(CHF₂)—; —CH₂CH(CH₂F)—; —CH₂CH(F)CH₂—; —CH₂C(F₂)CH₂—;—CH₂CH(C(O)NR²⁰R²¹)—; —CH₂CH(C(O)OR²²)—; —CH₂CH(C(O)OH)—;—CH(CH₂F)CH₂CH(CH₂F)—; —CH(CHF₂)CH₂CH(CHF₂)—; —CH(CF₃)CH₂CH(CF₃)—;—CH₂CH₂CH(CF₃)—; —CH₂CH₂CH(CHF₂)—; —CH₂CH₂CH(CH₂F)—; —CH₂CH₂CH(C(O)NR²³R²⁴)—; —CH₂CH₂CH(C(O)OR²⁵)—; and —CH₂CH₂CH(C(O)OH)—, in which R²⁰,R²¹, R²² and R²³ each independently represents hydrogen or (1-6C)alkyl,and R²⁴ and R²⁵ each independently represents (1-6C)alkyl.

In formula KC-(I), n can be an integer from 2 to 4. In certaininstances, n is two. In other instances, n is three. In other instances,n is four.

In formula KC-(I), R⁴ can be a residue of an L-amino acid selected fromalanine, arginine, asparagine, aspartic acid, cysteine, glycine,glutamine, glutamic acid, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine and valine, or a residue of an N-acyl derivative of any of saidamino acids; or a residue of a peptide composed of at least two L-aminoacid residues selected independently from alanine, arginine, asparagine,aspartic acid, cysteine, glycine, glutamine, glutamic acid, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine or a residue of an N-acylderivative thereof. Such a peptide can be from 2 to about 100 aminoacids in length. Examples of N-acyl derivatives include acetyl, benzoyl,malonyl, piperonyl or succinyl derivatives.

In certain instances, R⁴ is a residue of L-arginine or L-lysine, or aresidue of an N-acyl derivative of L-arginine or L-lysine.

In certain instances, in formula KC-(I), when p is greater than one,then the R⁴ adjacent to the nitrogen of —N(R³)(R⁴) is a residue ofL-arginine or L-lysine. In certain instances, when p is greater thanone, the R⁴ adjacent to the nitrogen of —N(R³)(R⁴) is a residue ofL-arginine or L-lysine and the first residue is joined to at least oneadditional L-amino acid residue selected independently from alanine,arginine, asparagine, aspartic acid, cysteine, glycine, glutamine,glutamic acid, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine andvaline. The terminal residue of the peptide can be an N-acyl derivativeof any of such L-amino acids. In certain instances R⁴ is a dipeptide oran N-acyl derivative thereof. In certain instances R is a tripeptide oran N-acyl derivative thereof.

In formula KC-(I), R⁴ is

In formula KC-(I), each R⁶ can be independently selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring.

In certain instances, in formula KC-(I), R⁶ is selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl. In certaininstances, R⁶ is selected from hydrogen, alkyl, substituted alkyl,arylalkyl, substituted arylalkyl, heteroarylalkyl, and substitutedheteroarylalkyl. In certain instances, R⁶ is hydrogen. In certaininstances, R⁶ is alkyl. In certain instances, R⁶ is substituted alkyl.In certain instances, R⁶ is arylalkyl or substituted arylalkyl. Incertain instances, R⁶ is heteroarylalkyl or substituted heteroarylalkyl.

In certain instances, R⁶ is a side chain of an amino acid, such asalanine, arginine, asparagine, aspartic acid, cysteine, glycine,glutamine, glutamic acid, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine or valine. In certain instances, R⁶ is a side chain of anL-amino acid, such as L-alanine, L-arginine, L-asparagine, L-asparticacid, L-cysteine, L-glycine, L-glutamine, L-glutamic acid, L-histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine,L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine or L-valine.

In certain instances, R⁶ is —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂.

In formula KC-(I), each W can be independently —NR⁸—, —O— or —S—. Incertain instances, W is —NR⁸—. In certain instances, W is —O—. Incertain instances, W is —S—.

In formula KC-(I), each R⁸ can be independently hydrogen, alkyl,substituted alkyl, aryl or substituted aryl, or optionally, each R⁶ andR⁸ independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring.

In certain instances, in formula KC-(I), R⁸ is hydrogen or alkyl. Incertain instances, R⁸ is hydrogen. In certain instances, R⁸ is alkyl. Incertain instances, R⁸ is aryl. In certain instances, R⁶ and R⁸independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring.

In formula KC-(I), p can be an integer from one to 100 and each R⁶ canbe selected independently from a side chain of any amino acid. Incertain instances, p is an integer from one to 50. In certain instances,p is an integer from one to 90, 80, 70, 60, 50, 40, 30, 20, or 10. Incertain instances, p is about 100. In certain instances, p is about 75.In certain instances, p is about 50. In certain instances, p is about25. In certain instances, p is about 20. In certain instances, p isabout 15. In certain instances, p is about 10. In certain instances, pis about 9. In certain instances, p is about 8. In certain instances, pis about 7. In certain instances, p is about 6. In certain instances, pis about 5. In certain instances, p is about 4. In certain instances, pis about 3. In certain instances, p is about 2. In certain instances, pis about one.

In certain instances, the R⁶ of R⁴ adjacent to the nitrogen of—N(R³)(R⁴) is —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, and anyadditional R⁶ can be a side chain of any amino acid independentlyselected from alanine, arginine, asparagine, aspartic acid, cysteine,glycine, glutamine, glutamic acid, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine or valine.

In formula KC-(I), R⁷ can be selected from hydrogen, alkyl, substitutedalkyl, acyl, substituted acyl, alkoxycarbonyl, substitutedalkoxycarbonyl, aryl, substituted aryl, arylalkyl, and substitutedarylalkyl.

In certain instances, R⁷ is hydrogen, alkyl, acyl, or substituted acyl.In certain instances, R⁷ is hydrogen. In certain instances, R⁷ is alkyl.In certain instances, R⁷ is acyl or substituted acyl. In certaininstances, R⁷ is acyl. In certain instances, R⁷ is substituted acyl. Incertain instances, R⁷ can be acetyl, benzoyl, malonyl, piperonyl orsuccinyl.

Formula KC-(II)

Compounds of formula KC-(II) are compounds of formula KC-(I) in which R⁵is selected from (1-6C) alkyl, (1-6C) substituted alkyl,—(CH₂)_(q)(C₆H₄)—COOH, —(CH₂)_(q)(C₆H₄)—COOCH₃, and—(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is an integer from one to 10; n is 2or 3; R³ is hydrogen; R⁴ is an L-amino acid or peptide, where thepeptide can be comprised of L-amino acids. In one of its compositionaspects, the present embodiments provide a compound of formula KC-(II):

wherein:

R^(a) is hydrogen or hydroxyl;

R⁵ is selected from (1-6C)alkyl, (1-6C) substituted alkyl,—(CH₂)_(q)(C₆H₄)—COOH, —(CH₂)_(q)(C₆H₄)—COOCH₃, and—(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is an integer from one to 10;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl or substituted cycloalkyl group, or two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n is 2 or 3;

R³ is hydrogen;

R⁴ is a residue of an L-amino acid selected from alanine, arginine,asparagine, aspartic acid, cysteine, glycine, glutamine, glutamic acid,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine and valine, or aresidue of an N-acyl derivative of any of said amino acids; or a residueof a peptide composed of at least two L-amino acid residues selectedindependently from alanine, arginine, asparagine, aspartic acid,cysteine, glycine, glutamine, glutamic acid, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine or a residue of an N-acyl derivativethereof.

In certain embodiments in Formula KC-(II), R⁴ is a residue of an L-aminoacid selected from arginine and lysine.

In certain instances, in formula KC-(II), when R⁴ is a peptidecomprising more than one amino acid, then the R⁴ adjacent to thenitrogen of —N(R³)(R⁴) is a residue of L-arginine or L-lysine. Incertain instances R⁴ is a dipeptide or an N-acyl derivative thereof. Incertain instances R⁴ is a tripeptide or an N-acyl derivative thereof.

In certain embodiments in Formula KC-(II), R⁴ is a residue of an N-acylderivative thereof. In certain instances, R⁴ is a residue of an N-acylderivative thereof, where the N-acyl derivative is substituted, such as,but not limited to, malonyl and succinyl.

Formulae KC-(III) to KC-(V)

The compositions of the present disclosure include compounds of formulaeKC-(III) to KC-(V) shown below. Pharmaceutical compositions and methodsof the present disclosure also contemplate compounds of formulaeKC-(III) to KC-(V).

Formula KC-(III)

In one of its composition aspects, the present embodiments provide acompound of formula KC-(IIIa):

wherein:

X represents a residue of a ketone-containing opioid, wherein thehydrogen atom of the corresponding enolic group of the ketone isreplaced by a covalent bond to —C(O)—NR⁵—(C(R¹)(R²))_(n)—NR³R⁴;

R⁵ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl, substituted cycloalkyl, aryl, or substituted aryl group, ortwo R² or R³ groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, form a cycloalkyl, substitutedcycloalkyl, aryl, or substituted aryl group;

n is an integer from 2 to 4;

R³ is hydrogen;

R⁴ is

each R⁶ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring;

each W is independently —NR⁸—, —O— or —S—;

each R⁸ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl and substituted aryl, or optionally, each R⁶ and R⁸independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring;

p is an integer from one to 100; and

R⁷ is selected from hydrogen, alkyl, substituted alkyl, acyl,substituted acyl, alkoxycarbonyl, substituted alkoxycarbonyl, aryl,substituted aryl, arylalkyl, and substituted arylalkyl;

or a salt, hydrate or solvate thereof.

In one of its composition aspects, the present embodiments provide acompound of formula KC-(IIIb):

wherein:

X represents a residue of a ketone-containing opioid, wherein thehydrogen atom of the corresponding enolic group of the ketone isreplaced by a covalent bond to —C(O)—NR⁵—(C(R¹)(R²))_(n)—NR³R⁴;

R⁵ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl or substituted cycloalkyl group, or two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n is an integer from 2 to 4;

R³ is hydrogen;

R⁴ is

each R⁶ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring;

each W is independently —NR⁸—, —O— or —S—;

each R⁸ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl and substituted aryl, or optionally, each R⁶ and R⁸independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring;

p is an integer from one to 100; and

R⁷ is selected from hydrogen, alkyl, substituted alkyl, acyl,substituted acyl, alkoxycarbonyl, substituted alkoxycarbonyl, aryl,substituted aryl, arylalkyl, and substituted arylalkyl;

or a salt, hydrate or solvate thereof.

Reference to formula KC-(III) is meant to include compounds of formulaKC-(IIIa) and KC-(IIIb).

In formula KC-(III), R⁵ can be selected from alkyl, substituted alkyl,arylalkyl, substituted arylalkyl, aryl and substituted aryl. In certaininstances, R⁵ is (1-6C)alkyl. In other instances, R⁵ is (1-4C)alkyl. Inother instances, R⁵ is methyl or ethyl. In other instances, R⁵ ismethyl. In certain instances, R⁵ is ethyl.

In certain instances, R⁵ is substituted alkyl. In certain instances, R⁵is an alkyl group substituted with a carboxylic group such as acarboxylic acid, carboxylic ester or carboxylic amide. In certaininstances, R⁵ is —(CH₂)_(n)—COOH, —(CH₂)_(n)—COOCH₃, or—(CH₂)_(n)—COOCH₂CH₃, wherein n is a number form one to 10. In certaininstances, R¹ is —(CH₂)₅—COOH, —(CH₂)₅—COOCH₃, or —(CH₂)₅—COOCH₂CH₃.

In certain instances, in formula KC-(III), R⁵ is arylalkyl orsubstituted arylalkyl. In certain instances, in formula KC-(III), R⁵ isarylalkyl. In certain instances, R⁵ is substituted arylalkyl. In certaininstances, R⁵ is an arylalkyl group substituted with a carboxylic groupsuch as a carboxylic acid, carboxylic ester or carboxylic amide. Incertain instances, R⁵ is —(CH₂)_(q)(C₆H₄)—COOH, —(CH₂)_(q)(C₆H₄)—COOCH₃,or —(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is an integer from one to 10. Incertain instances, R⁵ is —CH₂(C₆H₄)—COOH, —CH₂(C₆H₄)—COOCH₃, or —CH₂(C₆H₄)—COOCH₂CH₃.

In certain instances, in formula KC-(III), R⁵ is aryl. In certaininstances, R⁵ is substituted aryl. In certain instances, R⁵ is an arylgroup ortho, meta or para-substituted with a carboxylic group such as acarboxylic acid, carboxylic ester or carboxylic amide. In certaininstances, R⁵ is —(C₆H₄)—COOH, —(C₆H₄)—COOCH₃, or —(C₆H₄)—COOCH₂CH₃.

In formula KC-(III), each R¹ can be independently selected fromhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, acyl, andaminoacyl. In certain instances, R¹ is hydrogen or alkyl. In certaininstances, R¹ is hydrogen. In certain instances, R¹ is alkyl. In certaininstances, R¹ is acyl. In certain instances, R¹ is aminoacyl.

In formula KC-(III), each R² can be independently selected fromhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, acyl, andaminoacyl. In certain instances, R² is hydrogen or alkyl. In certaininstances, R² is hydrogen. In certain instances, R² is alkyl. In certaininstances, R² is acyl. In certain instances, R² is aminoacyl.

In certain instances, R¹ and R² are hydrogen. In certain instances, R¹and R² on the same carbon are both alkyl. In certain instances, R¹ andR² on the same carbon are methyl. In certain instances, R¹ and R² on thesame carbon are ethyl.

In certain instances, R¹ and R¹ which are vicinal are both alkyl and R²and R² which are vicinal are both hydrogen. In certain instances, R¹ andR¹ which are vicinal are both ethyl and R² and R² which are vicinal areboth hydrogen. In certain instances, R¹ and R¹ which are vicinal areboth methyl and R² and R² which are vicinal are both hydrogen.

In certain instances, in the chain of —[C(R¹)(R²)]_(n)— in FormulaKC-(III), not every carbon is substituted. In certain instances, in thechain of —[C(R¹)(R²)]_(n)—, there is a combination of different alkylsubstituents, such as methyl or ethyl.

In certain instances, one of R¹ and R² is methyl, ethyl or other alkyland R⁵ is alkyl. In certain instances, R¹ and R¹ which are vicinal areboth alkyl and R² and R² which are vicinal are both hydrogen and R⁵ isalkyl. In certain instances, R¹ and R¹ which are vicinal are both ethyland R² and R² which are vicinal are both hydrogen and R⁵ is alkyl. Incertain instances, R¹ and R¹ which are vicinal are both methyl and R²and R² which are vicinal are both hydrogen and R⁵ is alkyl.

In certain instances, one of R¹ and R² is methyl, ethyl or other alkyland R⁵ is substituted alkyl. In certain instances, one of R¹ and R² ismethyl, ethyl or other alkyl and R⁵ is an alkyl group substituted with acarboxylic group such as a carboxylic acid, carboxylic ester orcarboxylic amide. In certain instances, one of R¹ and R² is methyl,ethyl or other alkyl and R⁵ is —(CH₂)_(q)(C₆H₄)—COOH,—(CH₂)_(q)(C₆H₄)—COOCH₃, or —(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is aninteger from one to 10. In certain instances, one of R¹ and R² ismethyl, ethyl or other alkyl and R⁵ is an alkyl group substituted withcarboxamide.

In formula KC-(III), R¹ and R² together with the carbon to which theyare attached can form a cycloalkyl or substituted cycloalkyl group, ortwo R¹ or R² groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, can form a cycloalkyl or substitutedcycloalkyl group. In certain instances, R¹ and R² together with thecarbon to which they are attached can form a cycloalkyl group. Thus, incertain instances, R¹ and R² on the same carbon form a spirocycle. Incertain instances, R¹ and R² together with the carbon to which they areattached can form a substituted cycloalkyl group. In certain instances,two R¹ or R² groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, can form a cycloalkyl group. Incertain instances, two R¹ or R² groups on adjacent carbon atoms,together with the carbon atoms to which they are attached, can form asubstituted cycloalkyl group.

In certain instances, R¹ and R² together with the carbon to which theyare attached can form an aryl or substituted aryl group, or two R¹ or R²groups on adjacent carbon atoms, together with the carbon atoms to whichthey are attached, can form an aryl or substituted aryl group. Incertain instances, two R¹ or R² groups on adjacent carbon atoms,together with the carbon atoms to which they are attached, form a phenylring. In certain instances, two R¹ or R² groups on adjacent carbonatoms, together with the carbon atoms to which they are attached, form asubstituted phenyl ring. In certain instances, two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a naphthyl ring.

In certain instances, one of R¹ and R² is aminoacyl.

In certain instances, one or both of R¹ and R² is aminoacyl comprisingphenylenediamine.

In certain instances, one of R¹ and R² is

wherein each R¹⁰ is independently selected from hydrogen, alkyl,substituted alkyl, and acyl and R¹⁰ is alkyl or substituted alkyl. Incertain instances, at least one of R¹⁰ is acyl. In certain instances, atleast one of R¹⁰ is alkyl or substituted alkyl. In certain instances, atleast one of R¹⁰ is hydrogen. In certain instances, both of R¹⁰ arehydrogen.

In certain instances, one of R¹ and R² is

wherein R¹⁰ is hydrogen, alkyl, substituted alkyl, or acyl. In certaininstances, R¹⁰ is acyl. In certain instances, R¹⁰ is alkyl orsubstituted alkyl. In certain instances, R¹⁰ is hydrogen.

In certain instances, one of R¹ and R² is

wherein each R¹⁰ is independently hydrogen, alkyl, substituted alkyl, oracyl and b is a number from one to 5. In certain instances, one of R¹and R² is

wherein each R¹⁰ is independently hydrogen, alkyl, substituted alkyl, oracyl. In certain instances, one of R¹ and R² is

wherein R^(10a) is alkyl and each R¹⁰ is independently hydrogen, alkyl,substituted alkyl, or acyl.

In certain instances, one of R¹ and R² is

wherein R¹⁰ is independently hydrogen, alkyl, substituted alkyl, or acyland b is a number from one to 5. In certain instances, one of R¹ and R²is

wherein R¹⁰ is independently hydrogen, alkyl, substituted alkyl, oracyl.

In certain instances, one of R¹ and R² is an aminoacyl group, such as—C(O)NR^(10a)R^(10b) wherein each R^(10a) and R^(10b) is independentlyselected from hydrogen, alkyl, substituted alkyl, and acyl. In certaininstances, one of R¹ and R² is an aminoacyl group, such as—C(O)NR^(10a)R^(10b) wherein R^(10a) is an alkyl and R^(10b) issubstituted alkyl. In certain instances, one of R¹ and R² is anaminoacyl group, such as —C(O)NR^(10a)R^(10b), wherein R^(10a) is analkyl and R^(10b) is alkyl substituted with a carboxylic acid orcarboxyl ester. In certain instances, one of R¹ and R² is an aminoacylgroup, such as —C(O)NR^(10a)R^(10b), wherein R^(10a) is methyl andR^(10b) is alkyl substituted with a carboxylic acid or carboxyl ester.

In certain instances, R¹ or R² can modulate a rate of intramolecularcyclization. R¹ or R² can speed up a rate of intramolecular cyclization,when compared to the corresponding molecule where R¹ and R² are bothhydrogen. In certain instances, R¹ or R² comprise anelectron-withdrawing group or an electron-donating group. In certaininstances, R¹ or R² comprise an electron-withdrawing group. In certaininstances, R¹ or R² comprise an electron-donating group.

Atoms and groups capable of functioning as electron withdrawingsubstituents are well known in the field of organic chemistry. Theyinclude electronegative atoms and groups containing electronegativeatoms. Such groups function to lower the basicity or protonation stateof a nucleophilic nitrogen in the beta position via inductive withdrawalof electron density. Such groups can also be positioned on otherpositions along the alkylene chain. Examples include halogen atoms (forexample, a fluorine atom), acyl groups (for example an alkanoyl group,an aroyl group, a carboxyl group, an alkoxycarbonyl group, anaryloxycarbonyl group or an aminocarbonyl group (such as a carbamoyl,alkylaminocarbonyl, dialkylaminocarbonyl or arylaminocarbonyl group)),an oxo (═O) substituent, a nitrile group, a nitro group, ether groups(for example an alkoxy group) and phenyl groups bearing a substituent atthe ortho position, the para position or both the ortho and the parapositions, each substituent being selected independently from a halogenatom, a fluoroalkyl group (such as trifluoromethyl), a nitro group, acyano group and a carboxyl group. Each of the electron withdrawingsubstituents can be selected independently from these.

In certain instances, —[C(R¹)(R²)]_(n)— is selected from—CH(CH₂F)CH(CH₂F)—; —CH(CHF₂)CH(CHF₂)—; —CH(CF₃)CH(CF₃)—; —CH₂CH(CF₃)—;—CH₂CH(CHF₂)—; —CH₂CH(CH₂F)—; —CH₂CH(F)CH₂—; —CH₂C(F₂)CH₂—;—CH₂CH(C(O)NR²⁰R²¹)—; —CH₂CH(C(O)OR²²)—; —CH₂CH(C(O)OH)—;—CH(CH₂F)CH₂CH(CH₂F)—; —CH(CHF₂)CH₂CH(CHF₂)—; —CH(CF₃)CH₂CH(CF₃)—;—CH₂CH₂CH(CF₃)—; —CH₂CH₂CH(CHF₂)—; —CH₂CH₂CH(CH₂F)—; —CH₂CH₂CH(C(O)NR²³R²⁴)—; —CH₂CH₂CH(C(O)OR²⁵)—; and —CH₂CH₂CH(C(O)OH)—, in which R²⁰,R²¹, R²² and R²³ each independently represents hydrogen or (1-6C)alkyl,and R²⁴ and R²⁵ each independently represents (1-6C)alkyl.

In formula KC-(III), n can be an integer from 2 to 4. In certaininstances, n is two. In other instances, n is three. In other instances,n is four.

In formula KC-(III), R⁴ can be a residue of an L-amino acid selectedfrom alanine, arginine, asparagine, aspartic acid, cysteine, glycine,glutamine, glutamic acid, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine and valine, or a residue of an N-acyl derivative of any of saidamino acids; or a residue of a peptide composed of at least two L-aminoacid residues selected independently from alanine, arginine, asparagine,aspartic acid, cysteine, glycine, glutamine, glutamic acid, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine or a residue of an N-acylderivative thereof. Such a peptide can be from 2 to about 100 aminoacids in length. Examples of N-acyl derivatives include acetyl, benzoyl,malonyl, piperonyl or succinyl derivatives.

In certain instances, R⁴ is a residue of L-arginine or L-lysine, or aresidue of an N-acyl derivative of L-arginine or L-lysine.

In certain instances, in formula KC-(III), when p is greater than one,then the R⁴ adjacent to the nitrogen of —N(R³)(R⁴) is a residue ofL-arginine or L-lysine. In certain instances, when p is greater thanone, the R⁴ adjacent to the nitrogen of —N(R³)(R⁴) is a residue ofL-arginine or L-lysine and the first residue is joined to at least oneadditional L-amino acid residue selected independently from alanine,arginine, asparagine, aspartic acid, cysteine, glycine, glutamine,glutamic acid, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine andvaline. The terminal residue of the peptide can be an N-acyl derivativeof any of such amino acids. In certain instances R⁴ is a dipeptide or anN-acyl derivative thereof. In certain instances R is a tripeptide or anN-acyl derivative thereof.

In formula KC-(III), R⁴ is

In formula KC-(III), each R⁶ can be independently selected fromhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl, and substitutedheteroarylalkyl, or optionally, R⁶ and R⁷ together with the atoms towhich they are bonded form a cycloheteroalkyl or substitutedcycloheteroalkyl ring.

In certain instances, in formula KC-(III), R⁶ is selected from hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl. In certaininstances, R⁶ is selected from hydrogen, alkyl, substituted alkyl,arylalkyl, substituted arylalkyl, heteroarylalkyl, and substitutedheteroarylalkyl. In certain instances, R⁶ is hydrogen. In certaininstances, R⁶ is alkyl. In certain instances, R⁶ is substituted alkyl.In certain instances, R⁶ is arylalkyl or substituted arylalkyl. Incertain instances, R⁶ is heteroarylalkyl or substituted heteroarylalkyl.

In certain instances, R⁶ is a side chain of an amino acid, such asalanine, arginine, asparagine, aspartic acid, cysteine, glycine,glutamine, glutamic acid, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine or valine. In certain instances, R⁶ is a side chain of anL-amino acid, such as L-alanine, L-arginine, L-asparagine, L-asparticacid, L-cysteine, L-glycine, L-glutamine, L-glutamic acid, L-histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine,L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine or L-valine.

In certain instances, R⁶ is —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂.

In formula KC-(III), each W can be independently —NR⁸—, —O— or —S—. Incertain instances, W is —NR⁸—. In certain instances, W is —O—. Incertain instances, W is —S—.

In formula KC-(III), each R⁸ can be independently hydrogen, alkyl,substituted alkyl, aryl or substituted aryl, or optionally, each R⁶ andR⁸ independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring.

In certain instances, in formula KC-(III), R⁸ is hydrogen or alkyl. Incertain instances, R⁸ is hydrogen. In certain instances, R⁸ is alkyl. Incertain instances, R⁸ is aryl. In certain instances, R⁶ and R⁸independently together with the atoms to which they are bonded form acycloheteroalkyl or substituted cycloheteroalkyl ring.

In formula KC-(III), p can be an integer from one to 100 and each R⁶ canbe selected independently from a side chain of any amino acid. Incertain instances, p is an integer from one to 50. In certain instances,p is an integer from one to 90, 80, 70, 60, 50, 40, 30, 20, or 10. Incertain instances, p is about 100. In certain instances, p is about 75.In certain instances, p is about 50. In certain instances, p is about25. In certain instances, p is about 20. In certain instances, p isabout 15. In certain instances, p is about 10. In certain instances, pis about 9. In certain instances, p is about 8. In certain instances, pis about 7. In certain instances, p is about 6. In certain instances, pis about 5. In certain instances, p is about 4. In certain instances, pis about 3. In certain instances, p is about 2. In certain instances, pis about one.

In certain instances, the R⁶ of R⁴ adjacent to the nitrogen of—N(R³)(R⁴) is —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, and anyadditional R⁶ can be a side chain of any amino acid independentlyselected from alanine, arginine, asparagine, aspartic acid, cysteine,glycine, glutamine, glutamic acid, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine or valine.

In formula KC-(III), R⁷ can be selected from hydrogen, alkyl,substituted alkyl, acyl, substituted acyl, alkoxycarbonyl, substitutedalkoxycarbonyl, aryl, substituted aryl, arylalkyl, and substitutedarylalkyl.

In certain instances, R⁷ is hydrogen, alkyl, acyl, or substituted acyl.In certain instances, R⁷ is hydrogen. In certain instances, R⁷ is alkyl.In certain instances, R⁷ is acyl or substituted acyl. In certaininstances, R⁷ is acyl. In certain instances, R⁷ is substituted acyl. Incertain instances, R⁷ can be acetyl, benzoyl, malonyl, piperonyl orsuccinyl.

Formula KC-(IV)

Compounds of formula KC-(IV) are compounds of formula KC-(III) in whichR⁵ is selected from (1-6C) alkyl, (1-6C) substituted alkyl,—(CH₂)_(q)(C₆H₄)—COOH, —(CH₂)_(q)(C₆H₄)—COOCH₃, and—(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is an integer from one to 10; n is 2or 3; R³ is hydrogen; R⁴ is an L-amino acid or peptide, where thepeptide can be comprised of L-amino acids. In one of its compositionaspects, the present embodiments provide a compound of formula KC-(IV):

wherein:

X represents a residue of a ketone-containing opioid, wherein thehydrogen atom of the corresponding enolic group of the ketone isreplaced by a covalent bond to —C(O)—NR⁵—(C(R¹)(R²))_(n)—NR³R⁴;

R⁵ is selected from (1-6C)alkyl, (1-6C) substituted alkyl,—(CH₂)_(q)(C₆H₄)—COOH, —(CH₂)_(q)(C₆H₄)—COOCH₃, and—(CH₂)_(q)(C₆H₄)—COOCH₂CH₃, where q is an integer from one to 10;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl or substituted cycloalkyl group, or two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group;

n is 2 or 3;

R³ is hydrogen;

R⁴ is a residue of an L-amino acid selected from alanine, arginine,asparagine, aspartic acid, cysteine, glycine, glutamine, glutamic acid,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine and valine, or aresidue of an N-acyl derivative of any of said amino acids; or a residueof a peptide composed of at least two L-amino acid residues selectedindependently from alanine, arginine, asparagine, aspartic acid,cysteine, glycine, glutamine, glutamic acid, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine or a residue of an N-acyl derivativethereof;

or a salt, hydrate or solvate thereof.

In certain embodiments in Formula KC-(IV), R⁴ is a residue of an L-aminoacid selected from arginine and lysine.

In certain instances, in formula KC-(IV), when R⁴ is a peptidecomprising more than one amino acid, then the R⁴ adjacent to thenitrogen of —N(R³)(R⁴) is a residue of L-arginine or L-lysine. Incertain instances R⁴ is a dipeptide or an N-acyl derivative thereof. Incertain instances R⁴ is a tripeptide or an N-acyl derivative thereof.

In certain embodiments in Formula KC-(IV), R⁴ is a residue of an N-acylderivative thereof. In certain instances, R⁴ is a residue of an N-acylderivative thereof, where the N-acyl derivative is substituted, such as,but not limited to, malonyl and succinyl.

Formulae KC-(V)

Compounds of formula KC-(V) are compounds of formula KC-(III) in whichR⁴ is a trypsin-cleavable moiety.

In one of its composition aspects, the present embodiments provide acompound of formula KC-(V):

wherein:

X represents a residue of a ketone-containing opioid, wherein thehydrogen atom of the corresponding enolic group of the ketone isreplaced by a covalent bond to —C(O)—NR⁵—(C(R¹)(R²))_(n)—NR³R⁴;

R⁵ is selected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl;

each R¹ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

each R² is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, acyl, and aminoacyl;

or R¹ and R² together with the carbon to which they are attached form acycloalkyl, substituted cycloalkyl, aryl, or substituted aryl group, ortwo R¹ or R² groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, form a cycloalkyl, substitutedcycloalkyl, aryl, or substituted aryl group;

n is an integer from 2 to 4;

R³ is hydrogen;

R⁴ is a trypsin-cleavable moiety;

or a salt, hydrate or solvate thereof.

In formula KC-(V), R⁴ is a trypsin-cleavable moiety. A trypsin-cleavablemoiety is a structural moiety that is capable of being cleaved bytrypsin. In certain instances, a trypsin-cleavable moiety comprises acharged moiety that can fit into an active site of trypsin and is ableto orient the prodrug for cleavage at a scissile bond. For instance, thecharged moiety can be a basic moiety that exists as a charged moiety atphysiological pH.

In certain embodiments, in formula KC-(V), R⁴ is—C(O)—CH(R^(6a))—NH(R^(7a)), wherein R^(6a) represents a side chain ofan amino acid or a derivative of a side chain of an amino acid thateffects R⁴ to be a trypsin-cleavable moiety. A derivative refers to asubstance that has been altered from another substance by modification,partial substitution, homologation, truncation, or a change in oxidationstate.

For example, to form a trypsin-cleavable moiety, R^(6a) can include, butis not limited to, a side chain of lysine (such as L-lysine), arginine(such as L-arginine), homolysine, homoarginine, and ornithine. Othervalues for R⁴ include, but are not limited to, arginine mimics, argininehomologues, arginine truncates, arginine with varying oxidation states(for instance, metabolites), lysine mimics, lysine homologues, lysinetruncates, and lysine with varying oxidation states (for instance,metabolites). Examples of arginine and lysine mimics includearylguanidines, arylamidines (substituted benzamidines), benzylamines,and (bicyclo[2.2.2]octan-1-yl)methanamine and derivatives thereof.

In certain instances, in formula KC-(V), R^(6a) represents—CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂, the configuration of thecarbon atom to which R⁴ is attached corresponding with that in anL-amino acid.

In formula KC-(V), R^(7a) is selected from hydrogen, alkyl, substitutedalkyl, acyl, substituted acyl, alkoxycarbonyl, substitutedalkoxycarbonyl, aryl, substituted aryl, arylalkyl, and substitutedarylalkyl. In certain instances, R^(7a) is an amino acid or an N-acylderivative of an amino acid. In certain instances, R^(7a) is a peptideor N-acyl derivative of such a peptide, where the peptide comprises oneto 100 amino acids and where each amino acid can be selectedindependently. In certain instances, there are one to 50 amino acids inthe peptide. In certain instances, there are one to 90, 80, 70, 60, 50,40, 30, 20, or 10 amino acids in the peptide. In certain instances,there are about 100 amino acids in the peptide. In certain instances,there are about 75 amino acids in the peptide. In certain instances,there are about 50 amino acids in the peptide. In certain instances,there are about 25 amino acids in the peptide. In certain instances,there are about 20 amino acids in the peptide. In certain instances,there are about 15 amino acids in the peptide. In certain instances,there are about 10 amino acids in the peptide. In certain instances,there are about 9 amino acids in the peptide. In certain instances,there are about 8 amino acids in the peptide. In certain instances,there are about 7 amino acids in the peptide. In certain instances,there are about 6 amino acids in the peptide. In certain instances,there are about 5 amino acids in the peptide. In certain instances,there are about 4 amino acids in the peptide. In certain instances,there are about 3 amino acids in the peptide. In certain instances,there are about 2 amino acids in the peptide. In certain instances,there is about 1 amino acid in the peptide.

Particular compounds of interest, and salts or solvates or stereoisomersthereof, include:

oxycodone 6-(N-methyl-N-(2-N′-acetylarginylamino))ethylcarbamate

hydrocodone 6-(N-methyl-N-(2-N′-acetylarginylamino))ethylcarbamate

oxycodone 6-(N-methyl-N-(2-N′-malonylarginylamino))ethylcarbamate

oxycodone6-(N-5′-carboxypentyl-N-(2-N′-acetylarginylamino))ethylcarbamate

hydrocodone 6-(N-methyl-N-(2-N′-malonylarginylamino))ethylcarbamate

oxycodone6-(N-methyl-N-(2-N′-acetylarginylamino-2-(N-methyl-N-carboxymethyl-acetamido))ethylcarbamate

wherein the amino acid residue is of the L configuration.

The embodiments provide a pharmaceutical composition, which comprises acompound of general Formula KC-(I) to KC-(II), or a pharmaceuticallyacceptable salt thereof.

The embodiments provide a pharmaceutical composition, which comprises acompound of general Formulae KC-(III) to KC-(V), or a pharmaceuticallyacceptable salt thereof.

The embodiments provide a pharmaceutical composition, which comprises acompound disclosed herein other than a compound of general FormulaeKC-(I) to KC-(II), or a pharmaceutically acceptable salt thereof.

General Synthetic Procedures for Formulae KC-(I) to KC-(VI)

A representative synthesis for compounds of Formulae KC-(I) and KC-(II)is shown in the following schemes. Compounds of Formulae KC-(III) toKC-(VI) can also be synthesized by using the disclosed methods. Arepresentative synthesis for Compound KC203 is shown in Scheme KC-1. InScheme KC-1, the terms R¹, R², R⁵, and n are defined herein. The termsPG and PG² are amino protecting groups.

In Scheme KC-1, Compound KC200 is a commercially available startingmaterial. Alternatively, Compound KC200 can be synthesized via a varietyof different synthetic routes using commercially available startingmaterials and/or starting materials prepared by conventional syntheticmethods.

With continued reference to Scheme KC-1, Compound KC200 is protected atthe amino group to form Compound KC201, wherein PG¹ and PG² are aminoprotecting groups. Amino protecting groups can be found in T. W. Greeneand P. G. M. Wuts, “Protective Groups in Organic Synthesis”, Fourthedition, Wiley, New York 2006. Representative amino-protecting groupsinclude, but are not limited to, formyl groups; acyl groups, for examplealkanoyl groups, such as acetyl; alkoxycarbonyl groups, such astert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups, such asbenzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);arylmethyl groups, such as benzyl (Bn), trityl (Tr), and1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBS); and the like.

In certain embodiments, PG¹ and PG² are Boc groups. Conditions forforming Boc groups on Compound KC201 can be found in Greene and Wuts.One method is reaction of Compound KC200 with di-tert-butyl dicarbonate.The reaction can optionally be run in the presence of an activatingagent, such as DMAP.

With continued reference to Scheme KC-1, the carboxybenzyl group onCompound KC201 is deprotected to form Compound KC202. Conditions toremove the carboxybenzyl group can be found in Greene and Wuts. Methodsto remove the carboxybenzyl group include hydrogenolysis of CompoundKC201 or treatment of Compound KC201 with HBr. One method to remove thecarboxybenzyl group is reaction of Compound KC201 with hydrogen andpalladium.

With continued reference to Scheme KC-1, Compound KC202 is reacted withphosgene to form Compound KC203. Reaction with phosgene forms an acylchloride on the amino group of Compound KC202. Other reagents can act assubstitutes for phosgene, such as diphosgene or triphosgene.

A representative synthesis for Compound KC302 is shown in Scheme KC-2.In Scheme 2, the terms R^(a), R¹, R², R⁵, and n are defined herein. Theterms PG¹ and PG² are amino protecting groups.

In Scheme KC-2, Compound KC300 is a commercially available startingmaterial. Alternatively, Compound KC300 can be synthesized via a varietyof different synthetic routes using commercially available startingmaterials and/or starting materials prepared by conventional syntheticmethods.

With continued reference to Scheme KC-2, Compound KC300 is reacted withCompound KC203 to form Compound KC301. In this reaction, the enolate ofCompound KC300 reacts with the acyl chloride of Compound KC203 to form acarbamate.

With continued reference to Scheme KC-2, the protecting groups PG¹ andPG² are removed from Compound KC301 to form Compound KC302. Conditionsto remove amino groups can be found in Greene and Wuts. When PG¹ and PG²are Boc groups, the protecting groups can be removed with acidicconditions, such as treatment with trifluoroacetic acid.

A representative synthesis for Compound KC402 is shown in Scheme KC-3.In Scheme KC-3, the terms R^(a), R¹, R², R⁵, R⁶, R⁷ and n are definedherein. The term PG³ is an amino protecting group.

In Scheme KC-3, Compound KC400 is a commercially available startingmaterial. Alternatively, Compound KC400 can be synthesized via a varietyof different synthetic routes using commercially available startingmaterials and/or starting materials prepared by conventional syntheticmethods.

With continued reference to Scheme KC-3, Compound KC302 reacts withCompound KC400 to form Compound KC401 in a peptide coupling reaction. Apeptide coupling reaction typically employs a conventional peptidecoupling reagent and is conducted under conventional coupling reactionconditions, typically in the presence of a trialkylamine, such asethyldiisopropylamine or diisopropylethylamine (DIEA). Suitable couplingreagents for use include, by way of example, carbodiimides, such asethyl-3-(3-dimethylamino)propylcarbodiimide (EDC),dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and thelike, and other well-known coupling reagents, such asN,N′-carbonyldiimidazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline(EEDQ), benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),O-(7-azabenzotriazol-1-yl)-N,N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and the like. Optionally, well-known couplingpromoters, such N-hydroxysuccinimide, 1-hydroxybenzotriazole (HOBT),1-hydroxy-7-azabenzotriazole (HOAT), N,N-dimethylaminopyridine (DMAP)and the like, can be employed in this reaction. Typically, this couplingreaction is conducted at a temperature ranging from about 0° C. to about60° C. for about 1 to about 72 hours in an inert diluent, such as THF orDMF. In certain instances, Compound KC302 reacts with Compound KC400 toform Compound KC401 in the presence of HATU and DIEA in DMF.

With continued reference to Scheme KC-3, Compound KC401 is transformedinto Compound KC402 with removal of the amino protecting group andaddition of an R⁷ group. In certain cases, the amino protecting group isR⁷ and removal of the amino protecting group is optional.

As disclosed herein, representative amino-protecting groups include, butare not limited to, formyl groups; acyl groups, for example alkanoylgroups, such as acetyl; alkoxycarbonyl groups, such astert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups, such asbenzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);arylmethyl groups, such as benzyl (Bn), trityl (Tr), and1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBS); and the like. In certainembodiments, PG³ is a Boc group. When PG³ is a Boc group, the protectinggroup can be removed with acidic conditions, such as treatment withtrifluoroacetic acid.

In certain instances, the R⁷ group is added to Compound KC401.Conditions for addition of R⁷ depend on the identity of R⁷ and are knownto those skilled in the art. In certain instances R⁷ is an acyl group,such as acetyl, benzoyl, malonyl, piperonyl or succinyl.

N-Acyl derivatives of the compounds of formula KC-(I) may convenientlybe prepared by acylating a corresponding compound of formula KC-(I)using an appropriate acylating agent, for example an anhydride, such asacetic anhydride (to prepare an N-acetyl compound) or an acid halide.The reaction is conveniently performed in the presence of a non-reactivebase, for example a tertiary amine, such as triethylamine. Convenientsolvents include amides, such as dimethyl formamide. The temperature atwhich the reaction is performed is conveniently in the range of from 0to 100° C., such as at ambient temperature.

With continued reference to Scheme KC-3, removal of other protectinggroups can be performed if other protecting groups were used, such asprotecting groups present on the R⁶ moiety. Conditions for removal ofother protecting groups depend on the identity of the protecting groupand are known to those skilled in the art. The conditions can also befound in Greene and Wuts.

As described in more detail herein, the disclosure provides processesand intermediates useful for preparing compounds of the presentdisclosure or a salt or solvate or stereoisomer thereof. Accordingly,the present disclosure provides a process of preparing a compound of thepresent disclosure, the process involves:

contacting a compound of formula:

with a compound of formula

wherein PG¹ and PG² are amino protecting groups.

Accordingly and as described in more detail herein, the presentdisclosure provides a process of preparing a compound of the presentdisclosure, the process involves:

contacting a compound of formula:

with a compound of formula

wherein PG³ is an amino protecting group.

In one instance, the above process further involving the step of forminga salt of a compound of the present disclosure. Embodiments are directedto the other processes described herein; and to the product prepared byany of the processes described herein.

Trypsin Inhibitors

The enzyme capable of cleaving the enzymatically-cleavable moiety of aketone-modified opioid prodrug can be a protease. In certainembodiments, the enzyme is an enzyme located in the gastrointestinal(GI) tract, i.e., a gastrointestinal enzyme, or a GI enzyme. The enzymecan be a digestive enzyme such as a gastric, intestinal, pancreatic orbrush border enzyme or enzyme of GI microbial flora, such as thoseinvolved in peptide hydrolysis. Examples include a pepsin, such aspepsin A or pepsin B; a trypsin; a chymotrypsin; an elastase; acarboxypeptidase, such as carboxypeptidase A or carboxypeptidase B; anaminopeptidase, such as aminopeptidase N or aminopeptidase A; anendopeptidase; an exopeptidase; a dipeptidylaminopeptidase, such asdipeptidylaminopeptidase IV; a dipeptidase; a tripeptidase; or anenteropeptidase. In certain embodiments, the enzyme is a cytoplasmicprotease located on or in the GI brush border. In certain embodiments,the enzyme is trypsin. Accordingly, in certain embodiments, thecorresponding composition is administered orally to the patient.

The disclosure provides for a composition comprising a GI enzymeinhibitor. Such an inhibitor can inhibit at least one of any of the GIenzymes disclosed herein. An example of a GI enzyme inhibitor is aprotease inhibitor, such as a trypsin inhibitor.

As used herein, the term “trypsin inhibitor” refers to any agent capableof inhibiting the action of trypsin on a substrate. The term “trypsininhibitor” also encompasses salts of trypsin inhibitors. The ability ofan agent to inhibit trypsin can be measured using assays well known inthe art. For example, in a typical assay, one unit corresponds to theamount of inhibitor that reduces the trypsin activity by onebenzoyl-L-arginine ethyl ester unit (BAEE-U). One BAEE-U is the amountof enzyme that increases the absorbance at 253 nm by 0.001 per minute atpH 7.6 and 25° C. See, for example, K. Ozawa, M. Laskowski, 1966, J.Biol. Chem. 241, 3955 and Y. Birk, 1976, Meth. Enzymol. 45, 700. Incertain instances, a trypsin inhibitor can interact with an active siteof trypsin, such as the S1 pocket and the S3/4 pocket. The S1 pocket hasan aspartate residue which has affinity for a positively charged moiety.The S3/4 pocket is a hydrophobic pocket. The disclosure provides forspecific trypsin inhibitors and non-specific serine protease inhibitors.

There are many trypsin inhibitors known in the art, both those specificto trypsin and those that inhibit trypsin and other proteases such aschymotrypsin. The disclosure provides for trypsin inhibitors that areproteins, peptides, and small molecules. The disclosure provides fortrypsin inhibitors that are irreversible inhibitors or reversibleinhibitors. The disclosure provides for trypsin inhibitors that arecompetitive inhibitors, non-competitive inhibitors, or uncompetitiveinhibitors. The disclosure provides for natural, synthetic orsemi-synthetic trypsin inhibitors.

Trypsin inhibitors can be derived from a variety of animal or vegetablesources: for example, soybean, corn, lima and other beans, squash,sunflower, bovine and other animal pancreas and lung, chicken and turkeyegg white, soy-based infant formula, and mammalian blood. Trypsininhibitors can also be of microbial origin: for example, antipain; see,for example, H. Umezawa, 1976, Meth. Enzymol. 45, 678.

In one embodiment, the trypsin inhibitor is derived from soybean.Trypsin inhibitors derived from soybean (Glycine max) are readilyavailable and are considered to be safe for human consumption. Theyinclude, but are not limited to, SBTI, which inhibits trypsin, andBowman-Birk inhibitor, which inhibits trypsin and chymotrypsin. Suchtrypsin inhibitors are available, for example from Sigma-Aldrich, St.Louis, Mo., USA.

A trypsin inhibitor can be an arginine mimic or lysine mimic, eithernatural or synthetic compound. In certain embodiments, the trypsininhibitor is an arginine mimic or a lysine mimic, wherein the argininemimic or lysine mimic is a synthetic compound. As used herein, anarginine mimic or lysine mimic can include a compound capable of bindingto the P¹ pocket of trypsin and/or interfering with trypsin active sitefunction. The arginine or lysine mimic can be a cleavable ornon-cleavable moiety.

Examples of trypsin inhibitors, which are arginine mimics and/or lysinemimics, include, but not limited to, arylguanidine, benzamidine,3,4-dichloroisocoumarin, diisopropylfluorophosphate, gabexate mesylate,and phenylmethanesulfonyl fluoride, or substituted versions or analogsthereof. In certain embodiments, trypsin inhibitors comprise acovalently modifiable group, such as a chloroketone moiety, an aldehydemoiety, or an epoxide moiety. Other examples of trypsin inhibitors areaprotinin, camostat and pentamidine.

Other examples of trypsin inhibitors include compounds of formula:

wherein:

Q¹ is selected from —O-Q⁴ or -Q⁴-COOH, where Q⁴ is C₁-C₄ alkyl;

Q² is N or CH; and

Q³ is aryl or substituted aryl.

Certain trypsin inhibitors include compounds of formula:

wherein:

Q⁵ is —C(O)—COOH or —NH-Q⁶-Q⁷-SO₂—C₆H₅, where

Q⁶ is —(CH₂)_(p)—COOH;

Q⁷ is —(CH₂)_(r)—C₆H₅;

Q⁸ is NH;

n is a number from zero to two;

o is zero or one;

p is an integer from one to three; and

r is an integer from one to three.

Certain trypsin inhibitors include compounds of formula:

wherein:

Q⁵ is —C(O)—COOH or —NH-Q⁶-Q⁷-SO₂—C₆H₅, where

Q⁶ is —(CH₂)_(p)—COOH;

Q⁷ is —(CH₂)_(r)—C₆H₅; and

p is an integer from one to three; and

r is an integer from one to three.

Certain trypsin inhibitors include the following:

Compound 101

(S)-ethyl 4-(5-guanidino-2- (naphthalene-2- sulfonamido)pentanoyl)piperazine-1-carboxylate Compound 102

(S)-ethyl 4-(5-guanidino-2- (2,4,6- triisopropylphenylsulfonamido)pentanoyl)piperazine-1- carboxylate Compound 103

(S)-ethyl 1-(5-guanidino-2- (naphthalene-2- sulfonamido)pentanoyl)piperidine-4-carboxylate Compound 104

(S)-ethyl 1-(5-guanidino-2- (2,4,6- triisopropylphenylsulfonamido)pentanoyl)piperidine- 4-carboxylate Compound 105

(S)-6-(4-(5-guanidino-2- (naphthalene-2- sulfonamido)pentanoyl)piperazin-1-yl)-6-oxohexanoic acid Compound 106

4-aminobenzimidamide (also 4-aminobenzamidine) Compound 107

3-(4- carbamimidoylphenyl)-2- oxopropanoic acid Compound 108

(S)-5-(4- carbamimidoylbenzylamino)- 5-oxo-4-((R)-4-phenyl- 2-(phenylmethylsulfonamido) butanamido)pentanoic acid Compound 109

6- carbamimidoylnaphthalen- 2-yl 4- (diaminomethyleneamino) benzoateCompound 110

4,4′-(pentane-1,5- diylbis(oxy))dibenzimidamide

In certain embodiments, the trypsin inhibitor is SBTI, BBSI, Compound101, Compound 106, Compound 108, Compound 109, or Compound 110. Incertain embodiments, the trypsin inhibitor is camostat.

In certain embodiments, the trypsin inhibitor is a compound of formulaT-I:

wherein

A represents a group of the following formula:

-   -   R^(t9) and R^(t10) each represents independently a hydrogen atom        or a C₁₋₄ alkyl group, R^(t8) represents a group selected from        the following formulae:

wherein R^(t11), R^(t12) and R^(t13) each represents independently

(1) a hydrogen atom,

(2) a phenyl group,

(3) a C₁₋₄ alkyl group substituted by a phenyl group,

(4) a C₁₋₁₀ alkyl group,

(5) a C₁₋₁₀ alkoxyl group,

(6) a C₂₋₁₀ alkenyl group having 1 to 3 double bonds,

(7) a C₂₋₁₀ alkynyl group having 1 to 2 triple bonds,

(8) a group of formula: R^(t15)—C(O)XR^(t16)

-   -   wherein R^(t15) represents a single bond or a C₁₋₈ alkylene        group,    -   X represents an oxygen atom or an NH-group, and    -   R^(t16) represents a hydrogen atom, a C₁₋₄ alkyl group, a phenyl        group or a C₁₋₄ alkyl group substituted by a phenyl group, or

(9) a C₃₋₇ cycloalkyl group;

the structure

represents a 4-7 membered monocyclic hetero-ring containing 1 to 2nitrogen or oxygen atoms,

R^(t14) represents a hydrogen atom, a C₁₋₄ alkyl group substituted by aphenyl group or a group of formula: COOR^(t17), wherein R^(t17)represents a hydrogen atom, a C₁₋₄ alkyl group or a C₁₋₄ alkyl groupsubstituted by a phenyl group;

provided that R^(t11), R^(t12) and R^(t13) do not representsimultaneously hydrogen atoms;

or nontoxic salts, acid addition salts or hydrates thereof.

In certain embodiments, the trypsin inhibitor is a compound selectedfrom the following:

In certain embodiments, the trypsin inhibitor is a compound of formulaT-II:

wherein

X is NH;

n is zero or one; and

R^(t1) is selected from hydrogen, halogen, nitro, alkyl, substitutedalkyl, alkoxy, carboxyl, alkoxycarbonyl, acyl, aminoacyl, guanidine,amidino, carbamide, amino, substituted amino, hydroxyl, cyano and—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n2), wherein each m isindependently zero to 2; and R^(n1) and R^(n2) are independentlyselected from hydrogen and C₁₋₄ alkyl.

In certain embodiments, in formula T-II, R^(t1) is guanidino or amidino.

In certain embodiments, in formula T-II, R^(t1) is—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2), wherein m is one andR^(n1) and R^(n2) are methyl.

In certain embodiments, the trypsin inhibitor is a compound of formulaT-III:

In certain embodiments, the trypsin inhibitor is a compound of formulaT-III:

wherein

X is NH;

n is zero or one;

L^(t1) is selected from —C(O)—O—; —O—C(O)—; —O—(CH₂)_(m)—O—;—OCH₂—Ar^(t2)—CH₂O—; —C(O)—NR^(t3)—; and —NR^(t3)—C(O)—;

R^(t3) is selected from hydrogen, C₁₋₆ alkyl, and substituted C₁₋₆alkyl;

Ar^(t1) and Ar^(t2) are independently a substituted or unsubstitutedaryl group;

m is a number from 1 to 3; and

R^(t2) is selected from hydrogen, halogen, nitro, alkyl, substitutedalkyl, alkoxy, carboxyl, alkoxycarbonyl, acyl, aminoacyl, guanidine,amidino, carbamide, amino, substituted amino, hydroxyl, cyano and—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n2), wherein each m isindependently zero to 2; and R^(n1) and R^(n2) are independentlyselected from hydrogen and C₁₋₄ alkyl.

In certain embodiments, in formula T-III, R^(t2) is guanidino oramidino.

In certain embodiments, in formula T-III, R^(t2) is—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2) wherein m is one andR^(n1) and R^(n2) are methyl.

In certain embodiments, the trypsin inhibitor is a compound of formulaT-IV:

wherein

each X is NH;

each n is independently zero or one;

L^(t1) is selected from —C(O)—O—; —O—C(O)—; —O—(CH₂)_(m)—O—;—OCH₂—Ar^(t2)—CH₂O—; —C(O)—NR^(t3)—; and —NR^(t3)—C(O)—;

R^(t3) is selected from hydrogen, C₁₋₆ alkyl, and substituted C₁₋₆alkyl;

Ar^(t1) and Ar^(t2) are independently a substituted or unsubstitutedaryl group; and

m is a number from 1 to 3.

In certain embodiments, in formula T-IV, Ar^(t1) or Ar^(t2) is phenyl.

In certain embodiments, in formula T-IV, Ar^(t1) or Ar^(t2) is naphthyl.

In certain embodiments, the trypsin inhibitor is Compound 109.

In certain embodiments, the trypsin inhibitor is

In certain embodiments, the trypsin inhibitor is Compound 110 or abis-arylamidine variant thereof; see, for example, J. D. Geratz, M.C.-F.Cheng and R. R. Tidwell (1976) J Med. Chem. 19, 634-639.

It will be appreciated that the pharmaceutical composition according tothe embodiments may further comprise one or more other trypsininhibitors.

It is to be appreciated that the invention also includes inhibitors ofother enzymes involved in protein assimilation that can be used incombination with a prodrug disclosed herein comprising an amino acid ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, orvaline or variants thereof. An amino acid variant refers to an aminoacid that is modified from a naturally-occurring amino acid but stillcomprises an activity similar to that of as the naturally-occurringamino acid.

Combinations of Prodrug and Trypsin Inhibitor

As discussed above, the present disclosure provides pharmaceuticalcompositions which comprise a trypsin inhibitor and a ketone-modifiedopioid prodrug that contains a trypsin-cleavable moiety that, whencleaved, facilitates release of ketone-containing opioid. Examples ofcompositions containing a ketone-modified opioid prodrug and a trypsininhibitor are described below.

Combinations of Formulae KC-(I) to KC-(II) and Trypsin Inhibitor

The embodiments provide a pharmaceutical composition, which comprises atrypsin inhibitor and a compound of general Formulae KC-(I) to KC-(II),or a pharmaceutically acceptable salt thereof. The embodiments provide apharmaceutical composition, which comprises a compound of Formulae T-Ito T-VI and a compound of general Formulae KC-(I) to KC-(II), or apharmaceutically acceptable salt thereof. The embodiments provide apharmaceutical composition, which comprises Compound 109 and a compoundof general Formulae KC-(I) to KC-(II), or a pharmaceutically acceptablesalt thereof.

Certain embodiments provide for a combination of a compound of FormulaKC-(I) and a trypsin inhibitor, in which the ketone-containing opioid ofFormula KC-(I) and the trypsin inhibitor are shown in the followingtable. Certain embodiments provide for a combination of a compound ofFormula KC-(II) and a trypsin inhibitor, in which the ketone-containingopioid of Formula KC-(II) and the trypsin inhibitor are also shown inthe following table.

Prodrug of Formula KC-(I) Prodrug of Formula KC-(II) Having IndicatedOpioid; Having Indicated Opioid; and Trypsin Inhibitor and TrypsinInhibitor Oxycodone; Hydrocodone; Oxycodone; Hydrocodone; SBTI SBTI SBTISBTI Oxycodone; Hydrocodone; Oxycodone; Hydrocodone; BBSI BBSI BBSI BBSIOxycodone; Hydrocodone; Oxycodone; Hydrocodone; Compound 101 Compound101 Compound 101 Compound 101 Oxycodone; Hydrocodone; Oxycodone;Hydrocodone; Compound 106 Compound 106 Compound 106 Compound 106Oxycodone; Hydrocodone; Oxycodone; Hydrocodone; Compound 108 Compound108 Compound 108 Compound 108 Oxycodone; Hydrocodone; Oxycodone;Hydrocodone; Compound 109 Compound 109 Compound 109 Compound 109Oxycodone; Hydrocodone; Oxycodone; Hydrocodone; Compound 110 Compound110 Compound 110 Compound 110

Combinations of Formulae KC-(III) to KC-(V) and Trypsin Inhibitor

The embodiments provide a pharmaceutical composition, which comprises atrypsin inhibitor and a compound of general Formulae KC-(III) to KC-(V),or a pharmaceutically acceptable salt thereof. The embodiments provide apharmaceutical composition, which comprises a compound of Formulae T-Ito T-VI and a compound of general Formulae KC-(III) to KC-(V), or apharmaceutically acceptable salt thereof. The embodiments provide apharmaceutical composition, which comprises Compound 109 and a compoundof general Formulae KC-(III) to KC-(V), or a pharmaceutically acceptablesalt thereof.

The embodiments provide a pharmaceutical composition, which comprises atrypsin inhibitor and a compound disclosed herein other than a compoundof general Formulae KC-(I) to KC-(II), or a pharmaceutically acceptablesalt thereof.

Certain embodiments provide for a combination of a compound of FormulaKC-(III) and a trypsin inhibitor, in which the ketone-containing opioidof Formula KC-(III) and the trypsin inhibitor are shown in the tablebelow. Certain embodiments provide for a combination of a compound ofFormula KC-(IV) and a trypsin inhibitor, in which the ketone-containingopioid of Formula KC-(IV) and the trypsin inhibitor are shown in thetable below. Certain embodiments provide for a combination of a compoundof Formula KC-(V) and a trypsin inhibitor, in which theketone-containing opioid of Formula KC-(V) and the trypsin inhibitor areshown in the following table.

Prodrug of Formula KC-(III) Prodrug of Formula KC-(IV) Prodrug ofFormula KC-(V) Having Indicated Opioid; Having Indicated Opioid; HavingIndicated Opioid; and Trypsin Inhibitor and Trypsin Inhibitor andTrypsin Inhibitor Oxycodone; Hydrocodone; Oxycodone; Hydrocodone;Oxycodone; Hydrocodone; SBTI SBTI SBTI SBTI SBTI SBTI Oxycodone;Hydrocodone; Oxycodone; Hydrocodone; Oxycodone; Hydrocodone; BBSI BBSIBBSI BBSI BBSI BBSI Oxycodone; Hydrocodone; Oxycodone; Hydrocodone;Oxycodone; Hydrocodone; Compound Compound Compound Compound CompoundCompound 101 101 101 101 101 101 Oxycodone; Hydrocodone; Oxycodone;Hydrocodone; Oxycodone; Hydrocodone; Compound Compound Compound CompoundCompound Compound 106 106 106 106 106 106 Oxycodone; Hydrocodone;Oxycodone; Hydrocodone; Oxycodone; Hydrocodone; Compound CompoundCompound Compound Compound Compound 108 108 108 108 108 108 Oxycodone;Hydrocodone; Oxycodone; Hydrocodone; Oxycodone; Hydrocodone; CompoundCompound Compound Compound Compound Compound 109 109 109 109 109 109Oxycodone; Hydrocodone; Oxycodone; Hydrocodone; Oxycodone; Hydrocodone;Compound Compound Compound Compound Compound Compound 110 110 110 110110 110

Combinations of Compound KC-2 and Trypsin Inhibitor

Certain embodiments provide for a combination of Compound KC-2 and atrypsin inhibitor, in which the trypsin inhibitor is shown in thefollowing table.

Compound Trypsin inhibitor Compound KC-2 SBTI Compound KC-2 BBSICompound KC-2 Compound 101 Compound KC-2 Compound 106 Compound KC-2Compound 108 Compound KC-2 Compound 109 Compound KC-2 Compound 110

Combinations of Ketone-Modified Opioid Prodrugs and Other Drugs

The disclosure provides for a ketone-modified opioid prodrug and afurther prodrug or drug included in a pharmaceutical composition. Such aprodrug or drug would provide additional analgesia or other benefits.Examples include opioids, acetaminophen, non-steroidal anti-inflammatorydrugs (NSAIDs) and other analgesics. In one embodiment, an opioidagonist prodrug or drug would be combined with an opioid antagonistprodrug or drug. Other examples include drugs or prodrugs that havebenefits other than, or in addition to, analgesia. The embodimentsprovide a pharmaceutical composition, which comprises a ketone-modifiedopioid prodrug and acetaminophen and optionally comprises a trypsininhibitor. Also included are pharmaceutically acceptable salts thereof.

In certain embodiments, the ketone-modified opioid prodrug is a compoundof general Formulae KC-(I) to KC-(V).

In certain embodiments, the trypsin inhibitor is selected from SBTI,BBSI, Compound 101, Compound 106, Compound 108, Compound 109, andCompound 110. In certain embodiments, the trypsin inhibitor is camostat.

In certain embodiments, a pharmaceutical composition can comprise aketone-modified opioid prodrug, a non-opioid drug and at least oneopioid or opioid prodrug.

Pharmaceutical Compositions and Methods of Use

The pharmaceutical composition according to the embodiments can furthercomprise a pharmaceutically acceptable carrier. The composition isconveniently formulated in a form suitable for oral (including buccaland sublingual) administration, for example as a tablet, capsule, thinfilm, powder, suspension, solution, syrup, dispersion or emulsion. Thecomposition can contain components conventional in pharmaceuticalpreparations, e.g. one or more carriers, binders, lubricants, excipients(e.g., to impart controlled release characteristics), pH modifiers,sweeteners, bulking agents, coloring agents or further active agents.

Patients can be humans, and also other mammals, such as livestock, zooanimals and companion animals, such as a cat, dog or horse.

In another aspect, the embodiments provide a pharmaceutical compositionas described hereinabove for use in the treatment of pain. Thepharmaceutical composition according to the embodiments is useful, forexample, in the treatment of a patient suffering from, or at risk ofsuffering from pain. Accordingly, the present disclosure providesmethods of treating or preventing pain in a subject, the methodsinvolving administering to the subject a disclosed composition. Thepresent disclosure provides for a disclosed composition for use intherapy or prevention or as a medicament. The present disclosure alsoprovides the use of a disclosed composition for the manufacture of amedicament, especially for the manufacture of a medicament for thetreatment or prevention of pain.

The compositions of the present disclosure can be used in the treatmentor prevention of pain including, but not limited to include, acute pain,chronic pain, neuropathic pain, acute traumatic pain, arthritic pain,osteoarthritic pain, rheumatoid arthritic pain, muscular skeletal pain,post-dental surgical pain, dental pain, myofascial pain, cancer pain,visceral pain, diabetic pain, muscular pain, post-herpetic neuralgicpain, chronic pelvic pain, endometriosis pain, pelvic inflammatory painand child birth related pain. Acute pain includes, but is not limitedto, acute traumatic pain or post-surgical pain. Chronic pain includes,but is not limited to, neuropathic pain, arthritic pain, osteoarthriticpain, rheumatoid arthritic pain, muscular skeletal pain, dental pain,myofascial pain, cancer pain, diabetic pain, visceral pain, muscularpain, post-herpetic neuralgic pain, chronic pelvic pain, endometriosispain, pelvic inflammatory pain and back pain.

The present disclosure provides use of a ketone-modified opioid prodrugand a trypsin inhibitor in the treatment of pain. The present disclosureprovides use of a ketone-modified opioid prodrug and a trypsin inhibitorin the prevention of pain.

The present disclosure provides use of a ketone-modified opioid prodrugand a trypsin inhibitor in the manufacture of a medicament for treatmentof pain. The present disclosure provides use of a ketone-modified opioidprodrug and a trypsin inhibitor in the manufacture of a medicament forprevention of pain.

In another aspect, the embodiments provide a method of treating pain ina patient requiring treatment, which comprises administering aneffective amount of a pharmaceutical composition as describedhereinabove. In another aspect, the embodiments provides method ofpreventing pain in a patient requiring treatment, which comprisesadministering an effective amount of a pharmaceutical composition asdescribed hereinabove.

The amount of composition disclosed herein to be administered to apatient to be effective (i.e. to provide blood levels ofketone-containing opioid sufficient to be effective in the treatment orprophylaxis of pain) will depend upon the bioavailability of theparticular composition, the susceptibility of the particular compositionto enzyme activation in the gut, the amount and potency of trypsininhibitor present in the composition, as well as other factors, such asthe species, age, weight, sex, and condition of the patient, manner ofadministration and judgment of the prescribing physician. In general,the dose can be such that the ketone-modified opioid prodrug is in therange of from 0.01 milligrams per kilogram to 20 milligrams prodrug perkilogram (mg/kg) body weight. For example, a prodrug comprising aresidue of oxycodone or hydrocodone can be administered at a doseequivalent to administering free oxycodone or hydrocodone in the rangeof from 0.02 to 0.5 mg/kg body weight or 0.01 mg/kg to 10 mg/kg bodyweight or 0.01 to 2 mg/kg body weight. In one embodiment wherein thecomposition comprises an oxycodone or hydrocodone prodrug, thecomposition can be administered at a dose such that the level ofoxycodone or hydrocodone achieved in the blood is in the range of from0.5 ng/ml to 10 ng/ml.

The amount of a trypsin inhibitor to be administered to the patient tobe effective (i.e. to attenuate release of ketone-containing opioid whenadministration of a compound disclosed herein alone would lead tooverexposure of the ketone-containing opioid) will depend upon theeffective dose of the particular prodrug and the potency of theparticular inhibitor, as well as other factors, such as the species,age, weight, sex and condition of the patient, manner of administrationand judgment of the prescribing physician. In general, the dose ofinhibitor can be in the range of from 0.05 mg to 50 mg per mg of prodrugdisclosed herein. In a certain embodiment, the dose of inhibitor can bein the range of from 0.001 mg to 50 mg per mg of prodrug disclosedherein. In one embodiment, the dose of inhibitor can be in the range offrom 0.01 nanomoles to 100 micromoles per micromole of prodrug disclosedherein.

Dose Units of Prodrug and Inhibitor Having a Desired PharmacokineticProfile

The present disclosure provides dose units of prodrug and inhibitor thatcan provide for a desired pharmacokinetic (PK) profile. Dose units canprovide a modified PK profile compared to a reference PK profile asdisclosed herein. It will be appreciated that a modified PK profile canprovide for a modified pharmacodynamic (PD) profile. Ingestion ofmultiples of such a dose unit can also provide a desired PK profile.

Unless specifically stated otherwise, “dose unit” as used herein refersto a combination of a GI enzyme-cleavable prodrug (e.g.,trypsin-cleavable prodrug) and a GI enzyme inhibitor (e.g., a trypsininhibitor). A “single dose unit” is a single unit of a combination of aGI enzyme-cleavable prodrug (e.g., trypsin-cleavable prodrug) and a GIenzyme inhibitor (e.g., trypsin inhibitor), where the single dose unitprovide a therapeutically effective amount of drug (i.e., a sufficientamount of drug to effect a therapeutic effect, e.g., a dose within therespective drug's therapeutic window, or therapeutic range). “Multipledose units” or “multiples of a dose unit” or a “multiple of a dose unit”refers to at least two single dose units.

As used herein, a “PK profile” refers to a profile of drug concentrationin blood or plasma. Such a profile can be a relationship of drugconcentration over time (i.e., a “concentration-time PK profile”) or arelationship of drug concentration versus number of doses ingested(i.e., a “concentration-dose PK profile”.) A PK profile is characterizedby PK parameters.

As used herein, a “PK parameter” refers to a measure of drugconcentration in blood or plasma, such as: 1) “drug Cmax”, the maximumconcentration of drug achieved in blood or plasma; 2) “drug Tmax”, thetime elapsed following ingestion to achieve Cmax; and 3) “drugexposure”, the total concentration of drug present in blood or plasmaover a selected period of time, which can be measured using the areaunder the curve (AUC) of a time course of drug release over a selectedperiod of time (t). Modification of one or more PK parameters providesfor a modified PK profile.

For purposes of describing the features of dose units of the presentdisclosure, “PK parameter values” that define a PK profile include drugCmax (e.g., ketone-containing opioid Cmax), total drug exposure (e.g.,area under the curve) (e.g., ketone-containing opioid exposure) and1/(drug Tmax) (such that a decreased 1/Tmax is indicative of a delay inTmax relative to a reference Tmax) (e.g., 1/ketone-containing opioidTmax). Thus a decrease in a PK parameter value relative to a referencePK parameter value can indicate, for example, a decrease in drug Cmax, adecrease in drug exposure, and/or a delayed Tmax.

Dose units of the present disclosure can be adapted to provide for amodified PK profile, e.g., a PK profile that is different from thatachieved from dosing a given dose of prodrug in the absence of inhibitor(i.e., without inhibitor). For example, dose units can provide for atleast one of decreased drug Cmax, delayed drug Tmax and/or decreaseddrug exposure compared to ingestion of a dose of prodrug in the sameamount but in the absence of inhibitor. Such a modification is due tothe inclusion of an inhibitor in the dose unit.

As used herein, “a pharmacodynamic (PD) profile” refers to a profile ofthe efficacy of a drug in a patient (or subject or user), which ischaracterized by PD parameters. “PD parameters” include “drug Emax” (themaximum drug efficacy), “drug EC50” (the concentration of drug at 50% ofthe Emax), and side effects.

FIG. 1 is a schematic illustrating an example of the effect ofincreasing inhibitor concentrations upon the PK parameter drug Cmax fora fixed dose of prodrug. At low concentrations of inhibitor, there maybe no detectable effect on drug release, as illustrated by the plateauportion of the plot of drug Cmax (Y axis) versus inhibitor concentration(X axis). As inhibitor concentration increases, a concentration isreached at which drug release from prodrug is attenuated, causing adecrease in, or suppression of, drug Cmax. Thus, the effect of inhibitorupon a prodrug PK parameter for a dose unit of the present disclosurecan range from undetectable, to moderate, to complete inhibition (i.e.,no detectable drug release).

A dose unit can be adapted to provide for a desired PK profile (e.g., aconcentration-time PK profile) following ingestion of a single dose. Adose unit can be adapted to provide for a desired PK profile (e.g., aconcentration-dose PK profile) following ingestion of multiple doseunits (e.g., at least 2, at least 3, at least 4 or more dose units).

Dose Units Providing Modified PK Profiles

A combination of a prodrug and an inhibitor in a dose unit can provide adesired (or “pre-selected”) PK profile (e.g., a concentration-time PKprofile) following ingestion of a single dose.

The PK profile of such a dose unit can be characterized by one or moreof a pre-selected drug Cmax, a pre-selected drug Tmax or a pre-selecteddrug exposure. The PK profile of the dose unit can be modified comparedto a PK profile achieved from the equivalent dosage of prodrug in theabsence of inhibitor (i.e., a dose that is the same as the dose unitexcept that it lacks inhibitor).

A modified PK profile can have a decreased PK parameter value relativeto a reference PK parameter value (e.g., a PK parameter value of a PKprofile following ingestion of a dosage of prodrug that is equivalent toa dose unit except without inhibitor). For example, a dose unit canprovide for a decreased drug Cmax, decreased drug exposure, and/ordelayed drug Tmax.

FIG. 2 presents schematic graphs showing examples of modifiedconcentration-time PK profiles of a single dose unit. Panel A is aschematic of drug concentration in blood or plasma (Y axis) following aperiod of time (X axis) after ingestion of prodrug in the absence orpresence of inhibitor. The solid, top line in Panel A provides anexample of drug concentration following ingestion of prodrug withoutinhibitor. The dashed, lower line in Panel A represents drugconcentration following ingestion of the same dose of prodrug withinhibitor. Ingestion of inhibitor with prodrug provides for a decreaseddrug Cmax relative to the drug Cmax that results from ingestion of thesame amount of prodrug in the absence of inhibitor. Panel A alsoillustrates that the total drug exposure following ingestion of prodrugwith inhibitor is also decreased relative to ingestion of the sameamount of prodrug without inhibitor.

Panel B of FIG. 2 provides another example of a dose unit having amodified concentration-time PK profile. As in Panel A, the solid topline represents drug concentration over time in blood or plasmafollowing ingestion of prodrug without inhibitor, while the dashed lowerline represents drug concentration following ingestion of the sameamount of prodrug with inhibitor. In this example, the dose unitprovides a PK profile having a decreased drug Cmax, decreased drugexposure, and a delayed drug Tmax (i.e., decreased (1/drug Tmax)relative to ingestion of the same dose of prodrug without inhibitor.

Panel C of FIG. 2 provides another example of a dose unit having amodified concentration-time PK profile. As in Panel A, the solid linerepresents drug concentration over time in blood or plasma followingingestion of prodrug without inhibitor, while the dashed line representsdrug concentration following ingestion of the same amount of prodrugwith inhibitor. In this example, the dose unit provides a PK profilehaving a delayed drug Tmax (i.e., decreased (1/drug Tmax) relative toingestion of the same dose of prodrug without inhibitor.

Dose units that provide for a modified PK profile (e.g., a decreaseddrug Cmax and/or delayed drug Tmax as compared to, a PK profile of drugor a PK profile of prodrug without inhibitor), find use in tailoring ofdrug dose according to a patient's needs (e.g., through selection of aparticular dose unit and/or selection of a dosage regimen), reduction ofside effects, and/or improvement in patient compliance (as compared toside effects or patient compliance associated with drug or with prodrugwithout inhibitor). As used herein, “patient compliance” refers towhether a patient follows the direction of a clinician (e.g., aphysician) including ingestion of a dose that is neither significantlyabove nor significantly below that prescribed. Such dose units alsoreduce the risk of misuse, abuse or overdose by a patient as compared tosuch risk(s) associated with drug or prodrug without inhibitor. Forexample, dose units with a decreased drug Cmax provide less reward foringestion than does a dose of the same amount of drug, and/or the sameamount of prodrug without inhibitor.

Dose Units Providing Modified PK Profiles Upon Ingestion of MultipleDose Units

A dose unit of the present disclosure can be adapted to provide for adesired PK profile (e.g., a concentration-time PK profile orconcentration-dose PK profile) following ingestion of multiples of adose unit (e.g., at least 2, at least 3, at least 4, or more doseunits). A concentration-dose PK profile refers to the relationshipbetween a selected PK parameter and a number of single dose unitsingested. Such a profile can be dose proportional, linear (a linear PKprofile) or nonlinear (a nonlinear PK profile). A modifiedconcentration-dose PK profile can be provided by adjusting the relativeamounts of prodrug and inhibitor contained in a single dose unit and/orby using a different prodrug and/or inhibitor.

FIG. 3 provides schematics of examples of concentration-dose PK profiles(exemplified by drug Cmax, Y axis) that can be provided by ingestion ofmultiples of a dose unit (X axis) of the present disclosure. Eachprofile can be compared to a concentration-dose PK profile provided byincreasing doses of drug alone, where the amount of drug in the blood orplasma from one dose represents a therapeutically effective amountequivalent to the amount of drug released into the blood or plasma byone dose unit of the disclosure. Such a “drug alone” PK profile istypically dose proportional, having a forty-five degree angle positivelinear slope. It is also to be appreciated that a concentration-dose PKprofile resulting from ingestion of multiples of a dose unit of thedisclosure can also be compared to other references, such as aconcentration-dose PK profile provided by ingestion of an increasingnumber of doses of prodrug without inhibitor wherein the amount of drugreleased into the blood or plasma by a single dose of prodrug in theabsence of inhibitor represents a therapeutically effective amountequivalent to the amount of drug released into the blood or plasma byone dose unit of the disclosure.

As illustrated by the relationship between prodrug and inhibitorconcentration in FIG. 2, a dose unit can include inhibitor in an amountthat does not detectably affect drug release following ingestion.Ingestion of multiples of such a dose unit can provide aconcentration-dose PK profile such that the relationship between numberof dose units ingested and PK parameter value is linear with a positiveslope, which is similar to, for example, a dose proportional PK profileof increasing amounts of prodrug alone. Panel A of FIG. 3 depicts such aprofile. Dose units that provide a concentration-dose PK profile havingsuch an undetectable change in drug Cmax in vivo compared to the profileof prodrug alone can find use in thwarting enzyme conversion of prodrugfrom a dose unit that has sufficient inhibitor to reduce or prevent invitro cleavage of the enzyme-cleavable prodrug by its respective enzyme.

Panel B in FIG. 3 represents a concentration-dose PK profile such thatthe relationship between the number of dose units ingested and a PKparameter value is linear with positive slope, where the profileexhibits a reduced slope relative to panel A. Such a dose unit providesa profile having a decreased PK parameter value (e.g., drug Cmax)relative to a reference PK parameter value exhibiting doseproportionality.

Concentration-dose PK profiles following ingestion of multiples of adose unit can be non-linear. Panel C in FIG. 3 represents an example ofa non-linear, biphasic concentration-dose PK profile. In this example,the biphasic concentration-dose PK profile contains a first phase overwhich the concentration-dose PK profile has a positive rise, and then asecond phase over which the relationship between number of dose unitsingested and a PK parameter value (e.g., drug Cmax) is relatively flat(substantially linear with zero slope). For such a dose unit, forexample, drug Cmax can be increased for a selected number of dose units(e.g., 2, 3, or 4 dose units). However, ingestion of additional doseunits does not provide for a significant increase in drug Cmax.

Panel D in FIG. 3 represents another example of a non-linear, biphasicconcentration-dose PK profile. In this example, the biphasicconcentration-dose PK profile is characterized by a first phase overwhich the concentration-dose PK profile has a positive rise and a secondphase over which the relationship between number of dose units ingestedand a PK parameter value (e.g., drug Cmax) declines. Dose units thatprovide this concentration-dose PK profile provide for an increase indrug Cmax for a selected number of ingested dose units (e.g., 2, 3, or 4dose units). However, ingestion of further additional dose units doesnot provide for a significant increase in drug Cmax and instead providesfor decreased drug Cmax.

Panel E in FIG. 3 represents a concentration-dose PK profile in whichthe relationship between the number of dose units ingested and a PKparameter (e.g., drug Cmax) is linear with zero slope. Such dose unitsdo not provide for a significant increase or decrease in drug Cmax withingestion of multiples of dose units.

Panel F in FIG. 3 represents a concentration-dose PK profile in whichthe relationship between number of dose units ingested and a PKparameter value (e.g., drug Cmax) is linear with a negative slope. Thusdrug Cmax decreases as the number of dose units ingested increases.

Dose units that provide for concentration-dose PK profiles whenmultiples of a dose unit are ingested find use in tailoring of a dosageregimen to provide a therapeutic level of released drug while reducingthe risk of overdose, misuse, or abuse. Such reduction in risk can becompared to a reference, e.g., to administration of drug alone orprodrug alone. In one embodiment, risk is reduced compared toadministration of a drug or prodrug that provides a proportionalconcentration-dose PK profile. A dose unit that provides for aconcentration-dose PK profile can reduce the risk of patient overdosethrough inadvertent ingestion of dose units above a prescribed dosage.Such a dose unit can reduce the risk of patient misuse (e.g., throughself-medication). Such a dose unit can discourage abuse throughdeliberate ingestion of multiple dose units. For example, a dose unitthat provides for a biphasic concentration-dose PK profile can allow foran increase in drug release for a limited number of dose units ingested,after which an increase in drug release with ingestion of more doseunits is not realized. In another example, a dose unit that provides fora concentration-dose PK profile of zero slope can allow for retention ofa similar drug release profile regardless of the number of dose unitsingested.

Ingestion of multiples of a dose unit can provide for adjustment of a PKparameter value relative to that of ingestion of multiples of the samedose (either as drug alone or as a prodrug) in the absence of inhibitorsuch that, for example, ingestion of a selected number (e.g., 2, 3, 4 ormore) of a single dose unit provides for a decrease in a PK parametervalue compared to ingestion of the same number of doses in the absenceof inhibitor.

Pharmaceutical compositions include those having an inhibitor to providefor protection of a therapeutic compound from degradation in the GItract. Inhibitor can be combined with a drug (i.e., not a prodrug) toprovide for protection of the drug from degradation in the GI system.

In this example, the composition of inhibitor and drug provide for amodified PK profile by increasing a PK parameter. Inhibitor can also becombined with a prodrug that is susceptible to degradation by a GIenzyme and has a site of action outside the GI tract. In thiscomposition, the inhibitor protects ingested prodrug in the GI tractprior to its distribution outside the GI tract and cleavage at a desiredsite of action.

Methods Used to Define Relative Amounts of Prodrug and Inhibitor in aDose Unit

Dose units that provide for a desired PK profile, such as a desiredconcentration-time PK profile and/or a desired concentration-dose PKprofile, can be made by combining a prodrug and an inhibitor in a doseunit in relative amounts effective to provide for release of drug thatprovides for a desired drug PK profile following ingestion by a patient.

Prodrugs can be selected as suitable for use in a dose unit bydetermining the GI enzyme-mediated drug release competency of theprodrug. This can be accomplished in vitro, in vivo or ex vivo.

In vitro assays can be conducted by combining a prodrug with a GI enzyme(e.g., trypsin) in a reaction mixture. The GI enzyme can be provided inthe reaction mixture in an amount sufficient to catalyze cleavage of theprodrug. Assays are conducted under suitable conditions, and optionallymay be under conditions that mimic those found in a GI tract of asubject, e.g., human. “Prodrug conversion” refers to release of drugfrom prodrug. Prodrug conversion can be assessed by detecting a level ofa product of prodrug conversion (e.g., released drug) and/or bydetecting a level of prodrug that is maintained in the presence of theGI enzyme. Prodrug conversion can also be assessed by detecting the rateat which a product of prodrug conversion occurs or the rate at whichprodrug disappears. An increase in released drug, or a decrease inprodrug, indicate prodrug conversion has occurred. Prodrugs that exhibitan acceptable level of prodrug conversion in the presence of the GIenzyme within an acceptable period of time are suitable for use in adose unit in combination with an inhibitor of the GI enzyme that isshown to mediate prodrug conversion.

In vivo assays can assess the suitability of a prodrug for use in a doseunit by administration of the prodrug to an animal (e.g., a human ornon-human animal, e.g., rat, dog, pig, etc.). Such administration can beenteral (e.g., oral administration). Prodrug conversion can be detectedby, for example, detecting a product of prodrug conversion (e.g.,released drug or a metabolite of released drug) or detecting prodrug inblood or plasma of the animal at a desired time point(s) followingadministration.

Ex vivo assays, such as a gut loop or inverted gut loop assay, canassess the suitability of a prodrug for use in a dose unit by, forexample, administration of the prodrug to a ligated section of theintestine of an animal. Prodrug conversion can be detected by, forexample, detecting a product of prodrug conversion (e.g., released drugor a metabolite of released drug) or detecting prodrug in the ligatedgut loop of the animal at a desired time point(s) followingadministration.

Inhibitors are generally selected based on, for example, activity ininteracting with the GI enzyme(s) that mediate release of drug from aprodrug with which the inhibitor is to be co-dosed. Such assays can beconducted in the presence of enzyme either with or without prodrug.Inhibitors can also be selected according to properties such ashalf-life in the GI system, potency, avidity, affinity, molecular sizeand/or enzyme inhibition profile (e.g., steepness of inhibition curve inan enzyme activity assay, inhibition initiation rate). Inhibitors foruse in prodrug-inhibitor combinations can be selected through use of invitro, in vivo and/or ex vivo assays.

One embodiment is a method for identifying a prodrug and a GI enzymeinhibitor suitable for formulation in a dose unit wherein the methodcomprises combining a prodrug (e.g., a ketone-modified opioid prodrug),a GI enzyme inhibitor (e.g., a trypsin inhibitor), and a GI enzyme(e.g., trypsin) in a reaction mixture and detecting prodrug conversion.Such a combination is tested for an interaction between the prodrug,inhibitor and enzyme, i.e., tested to determine how the inhibitor willinteract with the enzyme that mediates enzymatically-controlled releaseof the drug from the prodrug. In one embodiment, a decrease in prodrugconversion in the presence of the GI enzyme inhibitor as compared toprodrug conversion in the absence of the GI enzyme inhibitor indicatesthe prodrug and GI enzyme inhibitor are suitable for formulation in adose unit. Such a method can be an in vitro assay.

One embodiment is a method for identifying a prodrug and a GI enzymeinhibitor suitable for formulation in a dose unit wherein the methodcomprises administering to an animal a prodrug and a GI enzyme inhibitorand detecting prodrug conversion. In one embodiment, a decrease inprodrug conversion in the presence of the GI enzyme inhibitor ascompared to prodrug conversion in the absence of the GI enzyme inhibitorindicates the prodrug and GI enzyme inhibitor are suitable forformulation in a dose unit. Such a method can be an in vivo assay; forexample, the prodrug and GI enzyme inhibitor can be administered orally.Such a method can also be an ex vivo assay; for example, the prodrug andGI enzyme inhibitor can be administered orally or to a tissue, such asan intestine, that is at least temporarily exposed. Detection can occurin the blood or plasma or respective tissue. As used herein, tissuerefers to the tissue itself and can also refer to contents within thetissue.

One embodiment is a method for identifying a prodrug and a GI enzymeinhibitor suitable for formulation in a dose unit wherein the methodcomprises administering a prodrug and a gastrointestinal (GI) enzymeinhibitor to an animal tissue that has removed from an animal anddetecting prodrug conversion. In one embodiment, a decrease in prodrugconversion in the presence of the GI enzyme inhibitor as compared toprodrug conversion in the absence of the GI enzyme inhibitor indicatesthe prodrug and GI enzyme inhibitor are suitable for formulation in adose unit.

In vitro assays can be conducted by combining a prodrug, an inhibitorand a GI enzyme in a reaction mixture. The GI enzyme can be provided inthe reaction mixture in an amount sufficient to catalyze cleavage of theprodrug, and assays conducted under suitable conditions, optionallyunder conditions that mimic those found in a GI tract of a subject,e.g., human. Prodrug conversion can be assessed by detecting a level ofa product of prodrug conversion (e.g., released drug) and/or bydetecting a level of prodrug maintained in the presence of the GIenzyme. Prodrug conversion can also be assessed by detecting the rate atwhich a product of prodrug conversion occurs or the rate at whichprodrug disappears. Prodrug conversion that is modified in the presenceof inhibitor as compared to a level of prodrug conversion in the absenceof inhibitor indicates the inhibitor is suitable for attenuation ofprodrug conversion and for use in a dose unit. Reaction mixtures havinga fixed amount of prodrug and increasing amounts of inhibitor, or afixed amount of inhibitor and increasing amounts of prodrug, can be usedto identify relative amounts of prodrug and inhibitor which provide fora desired modification of prodrug conversion.

In vivo assays can assess combinations of prodrugs and inhibitors byco-dosing of prodrug and inhibitor to an animal. Such co-dosing can beenteral. “Co-dosing” refers to administration of prodrug and inhibitoras separate doses or a combined dose (i.e., in the same formulation).Prodrug conversion can be detected by, for example, detecting a productof prodrug conversion (e.g., released drug or drug metabolite) ordetecting prodrug in blood or plasma of the animal at a desired timepoint(s) following administration. Combinations of prodrug and inhibitorcan be identified that provide for a prodrug conversion level thatyields a desired PK profile as compared to, for example, prodrug withoutinhibitor.

Combinations of relative amounts of prodrug and inhibitor that providefor a desired PK profile can be identified by dosing animals with afixed amount of prodrug and increasing amounts of inhibitor, or with afixed amount of inhibitor and increasing amounts of prodrug. One or morePK parameters can then be assessed, e.g., drug Cmax, drug Tmax, and drugexposure. Relative amounts of prodrug and inhibitor that provide for adesired PK profile are identified as amounts of prodrug and inhibitorfor use in a dose unit. The PK profile of the prodrug and inhibitorcombination can be, for example, characterized by a decreased PKparameter value relative to prodrug without inhibitor. A decrease in thePK parameter value of an inhibitor-to-prodrug combination (e.g., adecrease in drug Cmax, a decrease in 1/drug Tmax (i.e., a delay in drugTmax) or a decrease in drug exposure) relative to a corresponding PKparameter value following administration of prodrug without inhibitorcan be indicative of an inhibitor-to-prodrug combination that canprovide a desired PK profile. Assays can be conducted with differentrelative amounts of inhibitor and prodrug.

In vivo assays can be used to identify combinations of prodrug andinhibitor that provide for dose units that provide for a desiredconcentration-dose PK profile following ingestion of multiples of thedose unit (e.g., at least 2, at least 3, at least 4 or more). Ex vivoassays can be conducted by direct administration of prodrug andinhibitor into a tissue and/or its contents of an animal, such as theintestine, including by introduction by injection into the lumen of aligated intestine (e.g., a gut loop, or intestinal loop, assay, or aninverted gut assay). An ex vivo assay can also be conducted by excisinga tissue and/or its contents from an animal and introducing prodrug andinhibitor into such tissues and/or contents.

For example, a dose of prodrug that is desired for a single dose unit isselected (e.g., an amount that provides an efficacious plasma druglevel). A multiple of single dose units for which a relationship betweenthat multiple and a PK parameter to be tested is then selected. Forexample, if a concentration-dose PK profile is to be designed foringestion of 2, 3, 4, 5, 6, 7, 8, 9 or 10 dose units, then the amount ofprodrug equivalent to ingestion of that same number of dose units isdetermined (referred to as the “high dose”). The multiple of dose unitscan be selected based on the number of ingested pills at which drug Cmaxis modified relative to ingestion of the single dose unit. If, forexample, the profile is to provide for abuse deterrence, then a multipleof 10 can be selected, for example. A variety of different inhibitors(e.g., from a panel of inhibitors) can be tested using differentrelative amounts of inhibitor and prodrug. Assays can be used toidentify suitable combination(s) of inhibitor and prodrug to obtain asingle dose unit that is therapeutically effective, wherein such acombination, when ingested as a multiple of dose units, provides amodified PK parameter compared to ingestion of the same multiple of drugor prodrug alone (wherein a single dose of either drug or prodrug alonereleases into blood or plasma the same amount of drug as is released bya single dose unit).

Increasing amounts of inhibitor are then co-dosed to animals with thehigh dose of prodrug. The dose level of inhibitor that provides adesired drug Cmax following ingestion of the high dose of prodrug isidentified and the resultant inhibitor-to-prodrug ratio determined.

Prodrug and inhibitor are then co-dosed in amounts equivalent to theinhibitor-to-prodrug ratio that provided the desired result at the highdose of prodrug. The PK parameter value of interest (e.g., drug Cmax) isthen assessed. If a desired PK parameter value results followingingestion of the single dose unit equivalent, then single dose unitsthat provide for a desired concentration-dose PK profile are identified.For example, where a zero dose linear profile is desired, the drug Cmaxfollowing ingestion of a single dose unit does not increasesignificantly following ingestion of a multiple number of the singledose units.

Methods for Manufacturing, Formulating, and Packaging Dose Units

Dose units of the present disclosure can be made using manufacturingmethods available in the art and can be of a variety of forms suitablefor enteral (including oral, buccal and sublingual) administration, forexample as a tablet, capsule, thin film, powder, suspension, solution,syrup, dispersion or emulsion. The dose unit can contain componentsconventional in pharmaceutical preparations, e.g. one or more carriers,binders, lubricants, excipients (e.g., to impart controlled releasecharacteristics), pH modifiers, flavoring agents (e.g., sweeteners),bulking agents, coloring agents or further active agents. Dose units ofthe present disclosure can include can include an enteric coating orother component(s) to facilitate protection from stomach acid, wheredesired.

Dose units can be of any suitable size or shape. The dose unit can be ofany shape suitable for enteral administration, e.g., ellipsoid,lenticular, circular, rectangular, cylindrical, and the like.

Dose units provided as dry dose units can have a total weight of fromabout 1 microgram to about 1 gram, and can be from about 5 micrograms to1.5 grams, from about 50 micrograms to 1 gram, from about 100 microgramsto 1 gram, from 50 micrograms to 750 milligrams, and may be from about 1microgram to 2 grams.

Dose units can comprise components in any relative amounts. For example,dose units can be from about 0.1% to 99% by weight of active ingredients(i.e., prodrug and inhibitor) per total weight of dose unit (0.1% to 99%total combined weight of prodrug and inhibitor per total weight ofsingle dose unit). In some embodiments, dose units can be from 10% to50%, from 20% to 40%, or about 30% by weight of active ingredients pertotal weight dose unit.

Dose units can be provided in a variety of different forms andoptionally provided in a manner suitable for storage. For example, doseunits can be disposed within a container suitable for containing apharmaceutical composition. The container can be, for example, a bottle(e.g., with a closure device, such as a cap), a blister pack (e.g.,which can provide for enclosure of one or more dose units per blister),a vial, flexible packaging (e.g., sealed Mylar or plastic bags), anampule (for single dose units in solution), a dropper, thin film, a tubeand the like.

Containers can include a cap (e.g., screw cap) that is removablyconnected to the container over an opening through which the dose unitsdisposed within the container can be accessed.

Containers can include a seal which can serve as a tamper-evident and/ortamper-resistant element, which seal is disrupted upon access to a doseunit disposed within the container. Such seal elements can be, forexample, a frangible element that is broken or otherwise modified uponaccess to a dose unit disposed within the container. Examples of suchfrangible seal elements include a seal positioned over a containeropening such that access to a dose unit within the container requiresdisruption of the seal (e.g., by peeling and/or piercing the seal).Examples of frangible seal elements include a frangible ring disposedaround a container opening and in connection with a cap such that thering is broken upon opening of the cap to access the dose units in thecontainer.

Dry and liquid dose units can be placed in a container (e.g., bottle orpackage, e.g., a flexible bag) of a size and configuration adapted tomaintain stability of dose units over a period during which the doseunits are dispensed into a prescription. For example, containers can besized and configured to contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100or more single dry or liquid dose units. The containers can be sealed orresealable. The containers can packaged in a carton (e.g., for shipmentfrom a manufacturer to a pharmacy or other dispensary). Such cartons canbe boxes, tubes, or of other configuration, and may be made of anymaterial (e.g., cardboard, plastic, and the like). The packaging systemand/or containers disposed therein can have one or more affixed labels(e.g., to provide information such as lot number, dose unit type,manufacturer, and the like).

The container can include a moisture barrier and/or light barrier, e.g.,to facilitate maintenance of stability of the active ingredients in thedose units contained therein. Where the dose unit is a dry dose unit,the container can include a desiccant pack which is disposed within thecontainer. The container can be adapted to contain a single dose unit ormultiples of a dose unit. The container can include a dispensing controlmechanism, such as a lock out mechanism that facilitates maintenance ofdosing regimen.

The dose units can be provided in solid or semi-solid form, and can be adry dose unit. “Dry dose unit” refers to a dose unit that is in otherthan in a completely liquid form. Examples of dry dose units include,for example, tablets, capsules (e.g., solid capsules, capsulescontaining liquid), thin film, microparticles, granules, powder and thelike. Dose units can be provided as liquid dose units, where the doseunits can be provided as single or multiple doses of a formulationcontaining prodrug and inhibitor in liquid form. Single doses of a dryor liquid dose unit can be disposed within a sealed container, andsealed containers optionally provided in a packaging system, e.g., toprovide for a prescribed number of doses, to provide for shipment ofdose units, and the like.

Dose units can be formulated such that the prodrug and inhibitor arepresent in the same carrier, e.g., solubilized or suspended within thesame matrix. Alternatively, dose units can be composed of two or moreportions, where the prodrug and inhibitor can be provided in the same ordifferent portions, and can be provided in adjacent or non-adjacentportions.

Dose units can be provided in a container in which they are disposed,and may be provided as part of a packaging system (optionally withinstructions for use). For example, dose units containing differentamounts of prodrug can be provided in separate containers, whichcontainers can be disposed with in a larger container (e.g., tofacilitate protection of dose units for shipment). For example, one ormore dose units as described herein can be provided in separatecontainers, where dose units of different composition are provided inseparate containers, and the separate containers disposed within packagefor dispensing.

In another example, dose units can be provided in a double-chambereddispenser where a first chamber contains a prodrug formulation and asecond chamber contains an inhibitor formulation. The dispenser can beadapted to provide for mixing of a prodrug formulation and an inhibitorformulation prior to ingestion. For example, the two chambers of thedispenser can be separated by a removable wall (e.g., frangible wall)that is broken or removed prior to administration to allow mixing of theformulations of the two chambers. The first and second chambers canterminate into a dispensing outlet, optionally through a common chamber.The formulations can be provided in dry or liquid form, or a combinationthereof. For example, the formulation in the first chamber can be liquidand the formulation in the second chamber can be dry, both can be dry,or both can be liquid.

Dose units that provide for controlled release of prodrug, of inhibitor,or of both prodrug and inhibitor are contemplated by the presentdisclosure, where “controlled release” refers to release of one or bothof prodrug and inhibitor from the dose unit over a selected period oftime and/or in a pre-selected manner.

Methods of Use of Dose Units

Dose units are advantageous because they find use in methods to reduceside effects and/or improve tolerability of drugs to patients in needthereof by, for example, limiting a PK parameter as disclosed herein.The present disclosure thus provides methods to reduce side effects byadministering a dose unit of the present disclosure to a patient in needso as to provide for a reduction of side effects as compared to thoseassociated with administration of drug and/or as compared toadministration of prodrug without inhibitor. The present disclosure alsoprovides methods to improve tolerability of drugs by administering adose unit of the present disclosure to a patient in need so as toprovide for improvement in tolerability as compared to administration ofdrug and/or as compared to administration of prodrug without inhibitor.

Dose units find use in methods for increasing patient compliance of apatient with a therapy prescribed by a clinician, where such methodsinvolve directing administration of a dose unit described herein to apatient in need of therapy so as to provide for increased patientcompliance as compared to a therapy involving administration of drugand/or as compared to administrations of prodrug without inhibitor. Suchmethods can help increase the likelihood that a clinician-specifiedtherapy occurs as prescribed.

Dose units can provide for enhanced patient compliance and cliniciancontrol. For example, by limiting a PK parameter (e.g., such as drugCmax or drug exposure) when multiples (e.g., two or more, three or more,or four or more) dose units are ingested, a patient requiring a higherdose of drug must seek the assistance of a clinician. The dose units canprovide for control of the degree to which a patient can readily“self-medicate”, and further can provide for the patient to adjust doseto a dose within a permissible range. Dose units can provide for reducedside effects, by for example, providing for delivery of drug at anefficacious dose but with a modified PK profile over a period oftreatment, e.g., as defined by a decreased PK parameter (e.g., decreaseddrug Cmax, decreased drug exposure).

Dose units find use in methods to reduce the risk of unintended overdoseof drug that can follow ingestion of multiple doses taken at the sametime or over a short period of time. Such methods of the presentdisclosure can provide for reduction of risk of unintended overdose ascompared to risk of unintended overdose of drug and/or as compared torisk of unintended overdose of prodrug without inhibitor. Such methodsinvolve directing administration of a dosage described herein to apatient in need of drug released by conversion of the prodrug. Suchmethods can help avoid unintended overdosing due to intentional orunintentional misuse of the dose unit.

The present disclosure provides methods to reduce misuse and abuse of adrug, as well as to reduce risk of overdose, that can accompanyingestion of multiples of doses of a drug, e.g., ingested at the sametime. Such methods generally involve combining in a dose unit a prodrugand an inhibitor of a GI enzyme that mediates release of drug from theprodrug, where the inhibitor is present in the dose unit in an amounteffective to attenuate release of drug from the prodrug, e.g., followingingestion of multiples of dose units by a patient. Such methods providefor a modified concentration-dose PK profile while providingtherapeutically effective levels from a single dose unit, as directed bythe prescribing clinician. Such methods can provide for, for example,reduction of risks that can accompany misuse and/or abuse of a prodrug,particularly where conversion of the prodrug provides for release of anarcotic or other drug of abuse (e.g., opioid). For example, when theprodrug provides for release of a drug of abuse, dose units can providefor reduction of reward that can follow ingestion of multiples of doseunits of a drug of abuse.

Dose units can provide clinicians with enhanced flexibility inprescribing drug. For example, a clinician can prescribe a dosageregimen involving different dose strengths, which can involve two ormore different dose units of prodrug and inhibitor having differentrelative amounts of prodrug, different amounts of inhibitor, ordifferent amounts of both prodrug and inhibitor. Such different strengthdose units can provide for delivery of drug according to different PKparameters (e.g., drug exposure, drug Cmax, and the like as describedherein). For example, a first dose unit can provide for delivery of afirst dose of drug following ingestion, and a second dose unit canprovide for delivery of a second dose of drug following ingestion. Thefirst and second prodrug doses of the dose units can be differentstrengths, e.g., the second dose can be greater than the first dose. Aclinician can thus prescribe a collection of two or more, or three ormore dose units of different strengths, which can be accompanied byinstructions to facilitate a degree of self-medication, e.g., toincrease delivery of an opioid drug according to a patient's needs totreat pain.

Thwarting Tampering by Trypsin Mediated Release of Ketone-ContainingOpioid from Prodrugs

The disclosure provides for a composition comprising a compounddisclosed herein and a trypsin inhibitor that reduces drug abusepotential. A trypsin inhibitor can thwart the ability of a user to applytrypsin to effect the release of a ketone-containing opioid from theketone-containing opioid prodrug in vitro. For example, if an abuserattempts to incubate trypsin with a composition of the embodiments thatincludes a ketone-containing opioid prodrug and a trypsin inhibitor, thetrypsin inhibitor can reduce the action of the added trypsin, therebythwarting attempts to release ketone-containing opioid for purposes ofabuse.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Celsius, and pressure is ator near atmospheric. Standard abbreviations may be used.

Synthesis of Small Molecule Trypsin Inhibitors Example 1 Synthesis of(S)-ethyl4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 101)

Preparation 1 Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-pentanoyl]-piperazine-1-carboxylicacid tert-butyl ester (A)

To a solution of Fmoc-Arg(Pbf)-OH 1 (25.0 g, 38.5 mmol) in DMF (200 mL)at room temperature was added DIEA (13.41 mL, 77.1 mmol). After stirringat room temperature for 10 min, the reaction mixture was cooled to ˜5°C. To the reaction mixture was added HATU (16.11 g, 42.4 mmol) inportions and stirred for 20 min and a solution oftert-butyl-1-piperazine carboxylate (7.18 g, 38.5 mmol) in DMF (50 mL)was added dropwise. The reaction mixture was stirred at ˜5° C. for 5min. The mixture reaction was then allowed to warm to room temperatureand stirred for 2 h. Solvent was removed in vacuo and the residue wasdissolved in EtOAc (500 mL), washed with water (2×750 mL), 1% H₂SO₄ (300mL) and brine (750 mL). The organic layer was separated, dried overNa₂SO₄ and solvent removed in vacuo to a total volume of 100 mL.Compound A was taken to the next step as EtOAc solution (100 mL). LC-MS[M+H] 817.5 (C₄₃H₅₆N₆O₈S+H, calc: 817.4).

Preparation 2 Synthesis of4-[(S)-2-Amino-5-({amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-pentanoyl]-piperazine-1-carboxylicacid tert-butyl ester (B)

To a solution of compound A (46.2 mmol) in EtOAc (175 mL) at roomtemperature was added piperidine (4.57 mL, 46.2 mmol) and the reactionmixture was stirred for 18 h at room temperature. Next the solvent wasremoved in vacuo and the resulting residue dissolved in minimum amountof EtOAc (˜50 mL) and hexane (˜1 L) was added. The precipitated crudeproduct was filtered off and recrystallised again with EtOAc (˜30 mL)and hexane (˜750 mL). The precipitate was filtered off, washed withhexane and dried in vacuo to afford compound B (28.0 g, 46.2 mmol).LC-MS [M+H] 595.4 (C₂₈H₄₆N₆O₆S+H, calc: 595.3). Compound B was usedwithout further purification.

Preparation 3 Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazine-1-carboxylicacid tert-butyl ester (C)

To a solution of compound B (28.0 g, 46.2 mmol) in THF (250 mL) wasadded aqueous 1N NaOH (171 mL). The reaction mixture was cooled to ˜5°C., a solution of 2-naphthalene sulfonylchloride (26.19 g, 115.6 mmol)in THF (125 mL) was added dropwise. The reaction mixture was stirred at˜5° C. for 10 min, with stirring continued at room temperature for 2 h.The reaction mixture was diluted with EtOAc (1 L), washed with aqueous1N NaOH (1 L), water (1 L) and brine (1 L). The organic layer wasseparated, dried over Na₂SO₄ and removal of the solvent in vacuo toafford compound C (36.6 g, 46.2 mmol). LC-MS [M+H] 785.5(C₃₈H₅₂N₆O₈S₂+H, calc: 785.9). Compound C was used without furtherpurification.

Preparation 4 Synthesis of2,2,4,6,7-Pentamethyl-2,3-dihydro-benzofuran-5-sulfonic acid1-amino-1-[(S)-4-(naphthalene-2-sulfonylamino)-5-oxo-5-piperazin-1-yl-pentylamino]-meth-(E)-ylideneamide(D)

To a solution of compound C (36.6 g, 46.2 mmol) in dioxane (60 mL) wasadded 4M HCl in dioxane (58 mL) dropwise. The reaction mixture wasstirred at room temperature for 1.5 h. Et₂O (600 mL) was added to thereaction mixture, the precipitated product was filtered off, washed withEt₂O and finally dried in vacuo to afford compound D (34.5 g, 46.2mmol). LC-MS [M+H] 685.4 (C₃₃H₄₄N₆O₆S₂+H, calc: 685.9). Compound D wasused without further purification.

Preparation 5 Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazine-1-carboxylicacid ethyl ester (E)

To a solution of compound D (8.0 g, 11.1 mmol) in CHCl₃ (50 mL) wasadded DIEA (4.1 mL, 23.3 mmol) at room temperature and stirred for 15min. The mixture was cooled to ˜5° C., ethyl chloroformate (1.06 mL,11.1 mmol) was added dropwise. After stirring at room temperatureovernight (˜18 h), solvent removed in vacuo. The residue was dissolvedin MeOH (˜25 mL) and Et₂O (˜500 mL) was added. The precipitated crudeproduct was filtered off, washed with Et₂O and dried in vacuo to affordcompound E (8.5 g, 11.1 mmol). LC-MS [M+H] 757.6 (C₃₆H₄₈N₆O₈S₂+H, calc:757.9). Compound E was used without further purification.

Synthesis of (S)-ethyl4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 101)

A solution of 5% m-cresol/TFA (50 mL) was added to compound E (8.5 g,11.1 mmol) at room temperature. After stirring for 1 h, the reactionmixture was precipitated with Et₂O (500 mL). The precipitate wasfiltered and washed with Et₂O and dried in vacuo to afford the crudeproduct. The crude product was purified by preparative reverse phaseHPLC. [Column: VARIAN, LOAD & LOCK, L&L 4002-2, Packing: Microsorb100-10 C18, Injection, Volume: ˜15 mL×2, Injection flow rate: 20 mL/min,100% A, (water/0.1% TFA), Flow rate: 100 mL/min, Fraction: 30 Sec (50mL), Method: 0% B (MeCN/0.1% TFA)-60% B/60 min/100 mL/min/254 nm].Solvents were removed from pure fractions in vacuo. Trace of water wasremoved by co-evaporation with 2×i-PrOH (50 mL). The residue wasdissolved in a minimum amount of i-PrOH and product was precipitatedwith 2 M HCl in Et₂O. Product was filtered off and washed with Et₂O anddried in vacuo to afford Compound 101 as HCl salt 7 (3.78 g, 63% yield,99.4% purity). LC-MS [M+H] 505.4 (C₃₈H₅₂N₆O₈S₂+H, calc: 505.6).

Example 2 Synthesis of (S)-ethyl4-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 102)

Preparation 6 Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-tert-butoxycarbonylamino-pentanoyl]-piperazine-1-carboxylicacid ethyl ester (F)

To a solution of Boc-Arg(Pbf)-OH (13.3 g, 25.3 mmol) in DMF (10 mL) wasadded DIEA (22.0 mL, 126.5 mmol) at room temperature and stirred for 15min. The reaction mixture was then cooled to ˜5° C. and HATU (11.5 g,30.3 mmol) was added in portions and stirred for 30 min, followed by thedropwise addition of ethyl-1-piperazine carboxylate (4.0 g, 25.3 mmol)in DMF (30 mL). After 40 min, the reaction mixture was diluted withEtOAc (400 mL) and poured into H₂O (1 L). Extracted with EtOAc (2×400mL) and washed with H₂O (800 mL), 2% H₂SO₄ (500 mL), H₂O (2×800 mL) andbrine (800 mL). Organic layer was separated, dried over MgSO₄ andsolvent removed in vacuo. The resultant oily residue was dried in vacuoto afford compound F (16.4 g, 24.5 mmol) as foamy solid. LC-MS [M+H]667.2 (C₃₁H₅₀N₆O₈S+H, calc: 667.8). Compound F was used without furtherpurification.

Preparation 7 Synthesis of4-[(S)-2-Amino-5-({amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5sulfonylimino]-methyl}-amino)-pentanoyl]-piperazine-1-carboxylic acidethyl ester (G)

A solution of compound F (20.2 g, 30.2 mmol) in dichloromethane (90 mL)was treated with 4.0 N HCl in 1,4-dioxane (90 mL, 363.3 mmol) andstirred at room temperature for 2 h. Next most of the dichloromethane(˜90%) was removed in vacuo and Et₂O (˜1 L) was added. The resultantprecipitate was filtered off and washed with Et₂O and dried in vacuo toafford compound G (17.8 g, 30.2 mmol). LC-MS [M+H] 567.8 (C₂₆H₄₂N₆O₆S+H,calc: 567.8). Compound G was used without further purification.

Preparation 8 Synthesis of4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(2,4,6-triisopropyl-benzenesulfonylamino)-pentanoyl]-piperazine-1-carboxylicacid ethyl ester (H)

To a solution of compound G (1.0 g, 1.8 mmol) in THF (7 mL) was added3.1N aqueous NaOH (4.0 mL) and stirred for 5 min. The reaction mixturewas cooled to ˜5° C., and then a solution of tripsyl chloride addeddropwise (2.2 g, 7.3 mmol) in THF (5 mL) and stirred at room temperatureovernight (˜18 h). The reaction mixture was diluted with H₂O (130 mL),acidified with 2% H₂SO₄ (15 mL) and extracted with EtOAc (3×80 mL).Organic layer were combined and washed with H₂O (2×400 mL), saturatedNaHCO₃ (100 mL), H₂O (200 mL) and brine (200 mL). The organic layer wasseparated, dried over MgSO₄ and solvent removed in vacuo to afford (2.9g) of crude product. This was purified by normal phase flashchromatography (5-10% MeOH/DCM) to afford compound H (0.52 g, 1.0 mmol).LC-MS [M+H] 833.8 (C₄₁H₆₄N₆O₈S₂+H, calc: 834.1).

Synthesis of (S)-ethyl4-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperazine-1-carboxylate(Compound 102)

A solution of 5% m-cresol/TFA (40 mL) was added to compound H (3.73 g,3.32 mmol) at room temperature. After stirring for 45 min, solvents wereremoved in vacuo. Residue was dissolved in dichloromethane (100 mL),washed with H₂O (3×200 mL) and brine (200 mL). The organic layer wasseparated, dried over MgSO₄ and then the solvent removed in vacuo. Theresidue was dissolved in dichloromethane (˜5 mL) and then hexane (250mL) was added and a precipitate was formed. This was washed with hexaneand dried in vacuo to afford the crude product (1.95 g). The crudeproduct was purified by reverse phase HPLC [Column: VARIAN, LOAD & LOCK,L&L 4002-2, Packing: Microsorb 100-10 C18, Injection Volume: ˜15 mL,Injection flow rate: 20 mL/min, 100% A, (water/0.1% TFA), Flow rate: 100mL/min, Fraction: 30 Sec (50 mL), Method: 25% B (MeCN/0.1% TFA)/70% B/98min/100 mL/min/254 nm]. Solvents were removed from pure fractions invacuo. Trace of water was removed by co-evaporation with 2×i-PrOH (50mL). The residue was dissolved in a minimum amount of i-PrOH and productwas precipitated with 2 M HCl in Et₂O. Product was filtered off andwashed with Et₂O and dried in vacuo to afford the product as HCl salt ofCompound 102 (0.72 g, 35% yield, 99.8% purity). LC-MS [M+H] 581.6(C₂₈H₄₈N₆O₅S+H, calc: 581.7).

Example 3 Synthesis of (S)-ethyl1-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 103)

Preparation 9 Synthesis of 1-[boc-Arg(Pbf)]-piperidine-4-carboxylic acidethyl ester (I)

To a solution of Boc-Arg(Pbf)-OH (3.4 g, 6.36 mmol) and HATU (2.9 g,7.63 mmol) in DMF (15 mL) was added DIEA (7.4 mL, 42.4 mmol) and thereaction mixture was stirred for 10 min at room temperature. A solutionof ethyl isonipecotate (1.0 g, 6.36 mmol) in DMF (6 mL) was added to thereaction mixture dropwise. The reaction mixture was stirred at roomtemperature for 1 h, then diluted with EtOAc (150 mL) and poured intowater (500 mL). The product was extracted with EtOAc (2×100 mL). Theorganic layer was washed with aqueous 0.1 N HCl (200 mL), 2% aqueoussodium bicarbonate (200 mL), water (200 mL) and brine (200 mL). Theorganic layer was then dried over sodium sulfate, filtered, and thenevaporated in vacuo. The resultant oily product was dried in vacuoovernight to give compound I (3.7 g, 5.57 mmol) as a viscous solid.LC-MS [M+H] 666.5 (C₃₂H₅₁N₅O₈S+H, calc: 666.7). Compound I was usedwithout further purification.

Preparation 10 Synthesis of 1-[Arg(Pbf)]-piperidine-4-carboxylic acidethyl ester HCl salt (J)

To a solution of compound I (4.7 g, 7.07 mmol) in dichloromethane (25mL) was added 4N HCl in dioxane (25.0 mL, 84.84 mmol), and the reactionmixture was stirred at room temperature for 2 h. The reaction mixturewas concentrated in vacuo to ˜20 mL of solvent, and then diluted withdiethyl ether (250 mL) to produce a white fine precipitate. The reactionmixture was stirred for 1 h and the solid was washed with ether (50 mL)and dried in vacuo overnight to give compound J (4.3 g, 7.07 mmol) as afine powder. LC-MS [M+H] 566.5 (C₂₇H₄₃N₅O₆S+H, calc: 566.7). Compound Jwas used without further purification.

Preparation 11 Synthesis of1-[5(S)—(N′-Pbf-guanidino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperidine-4-carboxylicacid ethyl ester (K)

To a solution of compound J (1.1 g, 1.6 mmol) and NaOH (260 mg, 5.9mmol) in a mixture of THF (5 mL) and water (3 mL) was added a solutionof 2-naphthalosulfonyl chloride (0.91 g, 2.5 mmol) in THF (10 mL)dropwise with stirring at ˜5° C. The reaction mixture was stirred atroom temperature for 1 h, then diluted with water (5 mL). Aqueous 1N HCl(5 mL) was added to obtain pH˜3. Additional water was added (20 mL), andthe product was extracted with ethyl acetate (3×50 mL). The organiclayer was removed and then washed with 2% aqueous sodium bicarbonate (50mL), water (50 mL) and brine (50 mL). The extract was dried overanhydrous sodium sulfate, filtered, and was evaporated in vacuo. Theformed oily product was dried in vacuo overnight to give compound K (1.3g, 1.6 mmol) as an oily foaming solid. LC-MS [M+H] 756.5(C₃₇H₄₉N₅O₈S₂+H, calc: 756.7). Compound K was used without furtherpurification.

Synthesis of (S)-ethyl1-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 103)

To a flask, was added compound K (1.3 g, 1.6 mmol) and then treated with5% m-cresol/TFA (10 mL). The reaction mixture was stirred at roomtemperature for 1 h. Next, the reaction mixture was concentrated invacuo to a volume ˜5 mL. Diethyl ether (200 mL) was then added to theresidue, and formed fine white precipitate. The precipitate was filteredoff and washed with ether (2×25 mL). The resultant solid was dried invacuo overnight to give a crude material, which was purified bypreparative reverse phase HPLC. [Nanosyn-Pack Microsorb (100-10)C-18column (50×300 mm); flow rate: 100 mL/min; injection volume 12 mL(DMSO-water, 1:1, v/v); mobile phase A: 100% water, 0.1% TFA; mobilephase B: 100% ACN, 0.1% TFA; gradient elution from 25% B to 55% B in 90min, detection at 254 nm]. Fractions containing desired compound werecombined and concentrated in vacuo. The residue was dissolved in i-PrOH(50 mL) and evaporated in vacuo (repeated twice). The residue was nextdissolved in i-PrOH (5 mL) and treated with 2 N HCl/ether (100 mL, 200mmol) to give a white precipitate. It was dried in vacuo overnight togive Compound 103 (306 mg, 31% yield, 95.7% purity) as a white solid.LC-MS [M+H] 504.5 (C₂₄H₃₃N₅O₅S+H, calc: 504.6).

Example 4 Synthesis of (S)-ethyl1-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 104)

Preparation 12 Synthesis of1-[5(S)—(N′-Pbf-guanidino)-2-(2,4,6-triisopropyl-benzenesulfonylamino)-pentanoyl]-piperidine-4-carboxylicacid ethyl ester (N)

To a solution of compound J (1.0 g, 1.6 mmol) and NaOH (420.0 mg, 10.4mmol) in a mixture of THF (5 mL) and water (4 mL) was added a solutionof 2,4,6-triisopropyl-benzenesulfonyl chloride (2.4 g, 8.0 mmol)dropwise with stirring and maintained at ˜5° C. The reaction mixture wasthen stirred at room temperature for 1 h, monitoring the reactionprogress, then diluted with water (20 mL), and acidified with aqueous 1N HCl (5 mL) to pH ˜3. Additional water was added (30 mL), and theproduct was extracted with EtOAc (3×50 mL). The organic layer was washedwith 2% aqueous sodium bicarbonate (50 mL), water (50 mL) and brine (50mL). The organic layer was dried over anhydrous sodium sulfate,filtered, and was evaporated in vacuo. Formed oily residue was dried ina vacuo overnight to give compound N (1.0 g, 1.2 mmol) as an oilymaterial. LC-MS [M+H] 832.8 (C₄₂H₆₅N₅O₈S₂+H, calc: 832.7). Compound Nwas used without further purification.

Synthesis of (S)-ethyl1-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperidine-4-carboxylateHCl salt (Compound 104)

To a flask was added compound N (2.3 g, 2.8 mmol) and then treated with5% m-cresol/TFA (16 mL). The reaction mixture was stirred at roomtemperature for 1 h. The reaction mixture was then concentrated in vacuoto a volume of 5 mL. Hexane (200 mL) was added to the residue anddecanted off to give an oily precipitate. The product was purified bypreparative reverse phase HPLC. [Nanosyn-Pack Microsorb (100-10)C-18column (50×300 mm); flow rate: 100 mL/min; injection volume 15 mL(DMSO-water, 1:1, v/v); mobile phase A: 100% water, 0.1% TFA; mobilephase B: 100% ACN, 0.1% TFA; gradient elution from 35% B to 70% B in 90min, detection at 254 nm]. Fractions containing desired compound werecombined and concentrated in vacuo. The residue was dissolved in i-PrOH(100 mL) and evaporated in vacuo (repeated twice). The residue wasdissolved in i-PrOH (5 mL) and treated with 2 N HCl/ether (100 mL, 200mmol) to give an oily residue. It was dried in vacuo overnight to giveCompound 104 (1.08 g, 62.8%) as a viscous solid. LC-MS [M+H] 580.6(C₂₉H₄₉N₅O₅S+H, calc: 580.8).

Example 5 Synthesis of(S)-6-(4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazin-1-yl)-6-oxohexanoicacid (Compound 105)

Preparation 13 Synthesis of6-{4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazin-1-yl}-6-oxo-hexanoicacid methyl ester (0)

To a solution of compound D (1.5 g, 2.08 mmol) in CHCl₃ (50 mL) wasadded DIEA (1.21 mL, 4.16 mmol) followed by adipoyl chloride (0.83 mL,6.93 mmol) dropwise. The reaction mixture was stirred at roomtemperature overnight (˜18 h). Solvents were removed in vacuo and theresidue was dried in vacuo to afford the compound O (2.1 g, amountexceeded quantitative). LC-MS [M+H] 827.5 (C₄₀H₅₄N₆O₉S₂+H, calc: 827.3).Compound O was used without further purification.

Preparation 14 Synthesis of6-{4-[(S)-5-({Amino-[(E)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-(naphthalene-2-sulfonylamino)-pentanoyl]-piperazin-1-yl}-6-oxohexanoicacid (P)

To a solution of compound O (2.1 g, 2.08 mmol) in THF (5 mL), H₂O (5 mL)was added 2 M aq LiOH (6 mL). The reaction mixture was stirred at roomtemperature for 2 h. Solvents were removed in vacuo, then the residuewas dissolved in water (˜50 mL), acidified with saturated aqueous NaHSO₄(˜100 mL) and extracted with EtOAc (2×100 mL). The organic layer wasdried over Na₂SO₄ and removal of the solvent gave compound P (1.72 g,2.08 mmol). LC-MS [M+H] 813.5 (C₃₉H₅₂N₆O₉S₂+H, calc: 813.3). Compound Pwas used without further purification.

Synthesis of(S)-6-(4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazin-1-yl)-6-oxohexanoicacid (Compound 105)

A solution of 5% m-cresol/TFA (25 mL) was added to compound P (1.72 g,2.08 mmol) at room temperature. After stirring for 30 min, the reactionmixture was precipitated with addition of Et₂O (˜200 mL). Theprecipitate was filtered and washed with Et₂O and dried in vacuo toafford the crude product. The crude product was purified by preparativereverse phase HPLC [Column: VARIAN, LOAD & LOCK, L&L 4002-2, Packing:Microsorb 100-10 C18, Injection Volume: ˜25 mL, Injection flow rate: 20mL/min, 95% A, (water/0.1% TFA), Flow rate: 100 mL/min, Fraction: 30 Sec(50 mL), Method: 5% B (MeCN/0.1% TFA)/5 min/25% B/20 min/25% B/15min/50% B/25 min/100 mL/min/254 nm]. Solvents were removed from purefractions in vacuo. Trace amounts of water was removed by co-evaporationwith i-PrOH (25 mL) (repeated twice). The residue was dissolved in aminimum amount of i-PrOH, then 2 M HCl in Et₂O (˜50 mL) was added anddiluted with Et₂O (˜250 mL). Precipitate formed was filtered off andwashed with Et₂O and dried in vacuo to afford the product as HCl saltCompound 105 (0.74 g, 59% yield, 98.9% purity). LC-MS [M+H] 561.4(C₂₆H₃₆N₆O₆S+H, calc: 561.2).

Example 6 Synthesis of 3-(4-carbamimidoylphenyl)-2-oxopropanoic acid(Compound 107)

Compound 107, i.e., 3-(4-carbamimidoylphenyl)-2-oxopropanoic acid can beproduced using methods known to those skilled in the art, such as thatdescribed by Richter P et al, Pharmazie, 1977, 32, 216-220 andreferences contained within. The purity of Compound 107 used herein wasestimated to be 76%, an estimate due low UV absorbance of this compoundvia HPLC. Mass spec data: LC-MS [M+H] 207.0 (C₁₀H₁₀N₂O₃+H, calc: 207.1).

Example 7 Synthesis of(S)-5-(4-carbamimidoylbenzylamino)-5-oxo-4-((R)-4-phenyl-2-(phenylmethylsulfonamido)butanamido)pentanoicacid (Compound 108)

Preparation 15 Synthesis of(S)-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)-butyric acidbenzyl ester (Q)

A solution of Boc-Glu(OBzl)-OH (7.08 g, 21.0 mmol), BOP (9.72 g, 22.0mmol) and DIEA (12.18 mL, 70.0 mmol) in DMF (50 mL) was maintained atroom temperature for 20 min, followed by the addition of4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). Thereaction mixture was stirred at room temperature for an additional 1 hand diluted with EtOAc (500 mL). The obtained solution was extractedwith water (100 mL), 5% aq. NaHCO₃ (100 mL) and water (2×100 mL). Theorganic layer was dried over MgSO₄, evaporated and dried in vacuo toprovide compound Q (9.65 g, yield exceeded quantitative) as yellowishoil. LC-MS [M+H] 452.0 (C₂₅H₂₉N₃O₅+H, calc: 452.4). Compound Q was usedwithout further purification.

Preparation 16 Synthesis of(S)-4-tert-butoxycarbonylamino-4-[4-(N-hydroxycarbamimidoyl)-benzylcarbamoyl]-butyric acid benzyl ester (R)

A solution of compound Q (9.65 g, 20.0 mmol), hydroxylaminehydrochloride (2.10 g, 30.0 mmol) and DIEA (5.22 mL, 30.0 mmol) inethanol (abs., 150 mL) was refluxed for 6 h. The reaction mixture wasallowed to cool to room temperature and stirred for additional 16 h. Thesolvents were evaporated in vacuo. The resultant residue was dried invacuo to provide compound R (14.8 g, yield exceeded quantitative) asyellowish oil. LC-MS [M+H] 485.5 (C₂₅H₃₂N₄O₆+H, calc: 485.8). Compound Rwas used without further purification.

Preparation 17 Synthesis of(S)-4-tert-butoxycarbonylamino-4-[4-(N-acetylhydroxycarbamimidoyl)-benzylcarbamoyl]-butyric acid benzyl ester (S)

A solution of compound R (14.8 g, 20.0 mmol) and acetic anhydride (5.7mL, 60.0 mmol) in acetic acid (100 mL) was stirred at room temperaturefor 45 min, and then solvent was evaporated in vacuo. The resultantresidue was dissolved in EtOAc (300 mL) and extracted with water (2×75mL) and brine (75 mL). The organic layer was then dried over MgSO₄,evaporated and dried in vacuo to provide compound S (9.58 g, 18.2 mmol)as yellowish solid. LC-MS [M+H] 527.6 (C₂₇H₃₄N₄O₇+H, calc: 527.9).Compound S was used without further purification.

Preparation 18 Synthesis of(S)-4-[4-(N-acetylhydroxycarbamimidoyl)-benzyl carbamoyl]-butyric acidbenzyl ester (T)

Compound S (9.58 g, 18.2 mmol) was dissolved in 1,4-dioxane (50 mL) andtreated with 4 N HCl/dioxane (50 mL, 200 mmol) at room temperature for 1h. Next, the solvent was evaporated in vacuo. The resultant residue wastriturated with ether (200 mL). The obtained precipitate was filtrated,washed with ether (100 mL) and hexane (50 mL) and dried in vacuo toprovide compound T (9.64 g, yield exceeded quantitative) as off-whitesolid. LC-MS [M+H]426.9 (C₂₂H₂₆N₄O₅+H, calc: 427.3). Compound T was usedwithout further purification.

Preparation 19 Synthesis of(R)-4-phenyl-2-phenylmethanesulfonylamino-butyric acid (U)

A solution of D-homo-phenylalanine (10.0 g, 55.9 mmol) and NaOH (3.35 g,83.8 mmol) in a mixture of 1,4-dioxane (80 mL) and water (50 mL) wascooled to ˜5° C., followed by alternate addition of α-toluenesulfonylchloride (16.0 g, 83.8 mmol; 5 portions by 3.2 g) and 1.12 M NaOH (50mL, 55.9 mmol; 5 portions by 10 mL) maintaining pH>10. The reactionmixture was then acidified with 2% aq. H₂SO₄ to a pH of about pH 2. Theobtained solution was extracted with EtOAc (2×200 mL). The obtainedorganic layer was washed with water (3×75 mL), dried over MgSO₄ and thenthe solvent was evaporated in vacuo. The resultant residue was dried invacuo to provide compound U (12.6 g, 37.5 mmol) as white solid. LC-MS[M+H] 334.2 (C₁₇H₁₉NO₄S+H, calc: 333.4). Compound U was used withoutfurther purification.

Preparation 20 Synthesis of(S)-4-[4-(N-acetylhydroxycarbamimidoyl)-benzylcarbamoyl]-4-((R)-4-phenyl-2-phenylmethanesulfonylamino-butyrylamino)-butyricacid benzyl ester (V)

A solution of compound U (5.9 g, 17.8 mmol), compound T di-hydrochloride(18.0 mmol), BOP (8.65 g, 19.6 mmol) and DIEA (10.96 mL, 19.6 mmol) inDMF (250 mL) was stirred at room temperature for 2 h. The reactionmixture was then diluted with EtOAc (750 mL) and extracted with water(200 mL). The formed precipitate was filtrated, washed with EtOAc (200mL) and water (200 mL) and dried at room temperature overnight (˜18 h)to provide compound V (8.2 g, 11.0 mmol) as off-white solid. LC-MS [M+H]743.6 (C₃₉H₄₃N₅O₈S+H, calc: 743.9). Compound V was used without furtherpurification.

Synthesis of(S)-5-(4-carbamimidoylbenzylamino)-5-oxo-4-((R)-4-phenyl-2-(phenylmethylsulfonamido)butanamido)pentanoicacid (Compound 108)

Compound V (8.0 g, 10.77 mmol) was dissolved in acetic acid (700 mL)followed by the addition of Pd/C (5% wt, 3.0 g) as a suspension in water(50 mL). Reaction mixture was subjected to hydrogenation (Parrapparatus, 50 psi H₂) at room temperature for 3 h. The catalyst wasfiltered over a pad of Celite on sintered glass filter and washed withmethanol. Filtrate was evaporated in vacuo to provide Compound 108 ascolorless oil. LC-MS [M+H] 594.2 (C₃₀H₃₅N₅O₆S+H, calc: 594). Obtainedoil was dissolved in water (150 mL) and subjected to HPLC purification.[Nanosyn-Pack YMC-ODS-A (100-10)C-18 column (75×300 mm); flow rate: 250mL/min; injection volume 150 mL; mobile phase A: 100% water, 0.1% TFA;mobile phase B: 100% acetonitrile, 0.1% TFA; isocratic elution at 10% Bin 4 min., gradient elution to 24% B in 18 min, isocratic elution at 24%B in 20 min, gradient elution from 24% B to 58% B in 68 min; detectionat 254 nm]. Fractions containing desired compound were combined andconcentrated in vacuo. Residue was dissolved in i-PrOH (75 mL) andevaporated in vacuo (procedure was repeated twice) to provide Compound108 (4.5 g, 70% yield, 98.0% purity) as white solid. LC-MS [M+H] 594.2(C₃₀H₃₅N₅O₆S+H, calc: 594). Retention time*: 3.55 min. *—[ChromolithSpeedRod RP-18e C18 column (4.6×50 mm); flow rate 1.5 mL/min; mobilephase A: 0.1% TFA/water; mobile phase B 0.1% TFA/acetonitrile; gradientelution from 5% B to 100% B over 9.6 min, detection 254 nm]

Synthesis of Ketone-modified Opioid Prodrugs Example 8: Synthesis ofN,N-Bis(tert-butyl) N′-2-(chlorocarbonyl(methyl)amino)ethylcarbamate

Preparation 21: Synthesis of[2-(Benzyloxycarbonyl-methyl-amino)-ethyl]-dicarbamic acid tert-butylester (P-1)

2-(Aminoethyl)-methyl-carbamic acid benzyl ester (2.0 g, 9.6 mmol) wasdissolved in dichloroethene (DCE) (20 mL) at room temperature. Triethylamine (NEt₃) (1.40 mL, 11.5 mmol) was added, followed by di-tert-butyldicarbonate (BOC₂O) (10.5 g, 48 mmol) and dimethylaminopyridine (DMAP)(120 mg). The reaction mixture was stirred at room temperature undernitrogen (N₂) for 2 h and then heated at 60° C. for 16 h. The reactionmixture was then concentrated. The residue was purified by silica gelchromatography, using 4/1 hexanes/EtOAc, to give P-1 in 86% yield (3.4g, 8.3 mmol). MS: (m/z) calc: 408.2, observed (M+Na⁺) 431.9.

Preparation 22: Synthesis of N1,N1-bis-BOC—N2-methylethane-1,2-diamine(P-2)

P-1 (1.3 g, 3.18 mmol) was dissolved in methanol/EtOAc (10 mL/3 mLrespectively). The mixture was degassed and saturated with N₂. Palladiumon carbon (Pd/C) (330 mg, 5% on carbon) was added. The mixture wasshaken in a Parr hydrogenator flask (50 psi H₂) for 4 h. The mixture wasthen filtered through a celite pad and the filtrate was concentrated togive P-2 (1.08 g, yield exceeded quantative). P-2 was used withoutfurther purification.

Synthesis of N,N-Bis(tert-butyl)N′-2-(chlorocarbonyl(methyl)amino)ethylcarbamate (E-8)

P-2 (500 mg, 1.82 mmol) and NEt₃ (0.4 mL, 2.74 mmol) was mixed togetherin dichloromethane (4 mL). The mixture was added to a pre-chilled to 0°C. solution of phosgene (5.5 mL, 0.5 M in toluene). The reaction mixturewas stirred at 0° C. for 1 h, followed by dilution with ether (20 mL)and filtered through filter paper. The filtrate was concentrated andpassed through a short silica gel column (10 cm×3 cm), eluted with 3/1hexanes/EtOAc. The fractions were concentrated to giveN,N-Bis(tert-butyl) N′-2-(chlorocarbonyl(methyl)amino)ethylcarbamate(E-8) as a colorless solid in quantative yield (615 mg, 1.82 mmol). MS:(m/z) calc: 336.1, observed (M+Na⁺) 359.8.

Example 9: Synthesis of Oxycodone6-(N-methyl-N-(2-amino)ethylcarbamate-2TFA

Synthesis of oxycodone 6-(N-methyl-N-(2-amino)ethylcarbamate-2TFA

Oxycodone free base (6.5 g, 20.6 mmol) was dissolved in dry, degassedtetrahydrofuran (120 mL), and the mixture was cooled to ˜10° C. usingdry ice/acetone bath. Potassium bis(trimethylsilyl)amide (KHMDS) (103.0mL, 51.6 mmol, 0.5 M in toluene) was added via cannula. The mixture wasstirred under N₂ at below −5° C. for 30 min. N,N-Bis(tert-butyl)N′-2-(chlorocarbonyl(methyl)amino)ethylcarbamate (8.0 g, 23.7 mmol),(E-8) prepared as described in Example 8, in THF (30 mL) was then addedvia cannula over 15 min. The mixture was stirred at −5° C. for 30 min.Another portion of carbamoyl chloride (4.0 g, 11.9 mmol) in THF (10 mL)was added. The reaction was stirred at room temperature for 2 h. Sodiumbicarbonate (10 mL, sat. aq.) was added. The mixture was concentrated invacuo to half of its initial volume. EtOAc (50 mL) was added and layerswere separated. The organic phase was further washed with water (3×20mL), brine (40 mL) and then was concentrated. The residue was purifiedby silica gel chromatography, using DCM/MeOH (gradient 100/1 to 100/15)to afford a white foam in 55% yield (7.0 g, 13.4 mmol). This materialwas dissolved in a 1:1 mixture of DCM/trifluoroacetic acid (TFA) (20mL/20 mL) at room temperature and stirred for 1 h. The solution was thenconcentrated in vacuo to afford oxycodone6-(N-methyl-N-(2-amino)ethylcarbamate-2TFA as a thick oil (7.3 g, 11.4mmol, 99% purity). MS: (m/z) calc: 415.2, observed (M+H⁺) 416.5. Theoxycodone 6-(N-methyl-N-(2-amino)ethylcarbamate-2TFA (E-9) was usedwithout further purification.

Example 10: Synthesis of Oxycodone6-(N-methyl-N-(2-N′-acetylarginylamino)) ethylcarbamate (Compound KC-2)

Preparation 23: Synthesis of oxycodone6-(N-methyl-N-(2-N′—Boc-arginyl(Pbf)amino))ethylcarbamate (P-3)

Oxycodone 6-(N-methyl-N-(2-amino)ethylcarbamate—2TFA (7.3 g, 11.4 mmol),prepared as described in Example 9, was dissolved in dimethylformamide(DMF) (60 mL). Boc-Arg(Pbf)-OH (6.0 g, 11.4 mmol), HATU (4.75 g, 12.5mmol) and diisopropylethylamine (DIPEA) (6.0 mL, 34.4 mmol) were addedin this order. The reaction was stirred at room temperature for 2 h. Themixture was then concentrated in vacuo and the residue was partitionedbetween EtOAc/water (100 mL/60 mL). The organic layer was washed withwater (60 mL), brine (50 mL), dried over Na₂SO₄ and concentrated toafford crude P-3 (11.0 g). P-3 was used without further purification.

Preparation 24: Synthesis of oxycodone6-(N-methyl-N-(2-N′-acetylarginyl(Pbf)amino))ethylcarbamate (P-4)

P-3 (11.0 g), prepared as described above, was dissolved into dioxane(10 mL) and cooled to 0° C. A hydrochloric acid (HCl) solution indioxane (4 N, 30 mL) was added. The mixture was stirred at roomtemperature for 3 h and then concentrated in vacuo. 10 g of the crudemixture was dissolved in a mixture of DIPEA (5.0 mL 28.5 mmol) in DCM(60 mL). Acetic anhydride (1.4 mL, 14.3 mmol) was added drop wise. Thereaction mixture was stirred at room temperature for 2 h. NaHCO₃ (30 mL,sat. aq.) was then added. The layers were separated and the DCM layerwas dried over Na₂SO₄, filtered and concentrated to afford P-4 (8.5 g).P-4 was used without further purification.

Synthesis of oxycodone6-(N-methyl-N-(2-N′-acetylarginylamino))ethylcarbamate, as the bis-TFAsalt (Compound KC-2)

P-4 (8.5 g) was dissolved in a mixture of m-cresol (3 mL) in TFA (30mL). The mixture was stirred at room temperature for 3 h. TFA was thenremoved in vacuo. The residue was dissolved into MeOH (10 mL) and addeddrop wise to a stirred HCl solution in ether (40 mL, 2 M). The whitesolid was filtered and washed with ethyl ether (4×30 mL). The whitesolid was further purified by prep HPLC (*RP-18e C18 column (4.6×50 mm);flow rate 1.5 mL/min; mobile phase A: 0.1% TFA/water; mobile phase B0.1% TFA/acetonitrile (CH₃CN); gradient elution), yielding Compound KC-2(3.5 g, 4.1 mmol, 96.6% purity). MS: (m/z) calc: 613.7, observed (M+H⁺)614.5.

Example 11: Synthesis ofN—{(S)-4-guanidino-1-[2-(methyl-[(5R,9R,13S,14S)-4,5a-epoxy-6,7-didehydro-14-hydroxy-3-methoxy-17-methylmorphinan-6-oxy]carbonyl-amino)-ethylcarbamoyl]-butyl}-malonamicacid (Compound KC-3)

Preparation 25: Synthesis of2,2,2-trifluoro-N-(2-methylamino-ethyl)-acetamide (A)

A solution of N-methylethylenediamine (27.0 g, 364 mmol) and ethyltrifluoroacetate (96.6 mL, 812 mmol) in a mixture of ACN (350 mL) andwater (7.8 mL, 436 mmol) was refluxed with stirring overnight. Solventswere evaporated in vacuo. The residue was re-evaporated with i-PrOH(3×100 mL), followed by heat-cool crystallization from DCM (500 mL).Formed crystals were filtered, washed with DCM and dried in vacuo toprovide compound A (88.3 g, 85%) as white solid powder.

Preparation 26: Synthesis ofmethyl-[2-(2,2,2-trifluoro-acetylamino)-ethyl]-carbamic acid benzylester (B)

A solution of compound A (88.2 g, 311 mmol) and DIEA (54.1 mL, 311 mmol)in THF (350 mL) was cooled in an ice bath, followed by the addition of asolution of N-(benzyloxycarbonyl)succinimide (76.6 g, 307 mmol) in THF(150 mL) drop wise over the period of 20 min. The temperature of thereaction mixture was raised to ambient temperature and stirring wascontinued for an additional 30 min. Solvents were then evaporated andthe resulting residue was dissolved in EtOAc (600 mL). The organic layerwas extracted with 5% aq. NaHCO₃ (2×150 mL) and brine (150 mL). Theorganic layer was evaporated to provide compound B as yellowish oil.LC-MS [M+H] 305.1 (C₁₃H₁₅F₃N₂O₃+H, calc: 305.3). Compound B was useddirectly in the next reaction without purification as a MeOH solution.

Preparation 27: Synthesis of (2-amino-ethyl)-methyl-carbamic acid benzylester (C)

To a solution of compound B (˜311 mmol) in MeOH (1.2 L) was added asolution of LiOH (14.9 g, 622 mmol) in water (120 mL). The reactionmixture was stirred at ambient temperature for 3 h. Solvents wereevaporated to 75% of the initial volume followed by dilution with water(400 mL). The solution was extracted with EtOAc (2×300 mL). The organiclayer was washed with brine (200 mL), dried over MgSO₄ and evaporated invacuo. The residue was dissolved in ether (300 mL) and treated with 2 NHCl/ether (200 mL). Formed precipitate was filtrated, washed with etherand dried in vacuo to provide the hydrochloric salt of compound C (67.8g, 89%) as a white solid. LC-MS [M+H] 209.0 (C₁₁H₁₆N₂O₂+H, calc: 209.3).Compound C was used directly in the next reaction without purificationas a DMF solution.

Preparation 28: Synthesis of{2-[boc-Arg(Pbf)]-aminoethyl}-methyl-carbamic acid benzyl ester (D)

A solution of Boc-Arg(Pbf)-OH (16.0 g, ˜30.4 mmol), compound Chydrochloride (8.2 g, 33.4 mmol) and DIEA (16.9 mL, 97.2 mmol) in DMF(150 mL) was cooled in an ice bath followed by the addition of asolution of HATU (13.8 g, 36.4 mmol) drop wise over 20 min. Thetemperature of the reaction mixture was raised to ambient temperatureand stirring was continued for an additional 1 h. The reaction mixturewas diluted with EtOAc (1 L) and extracted with water (3×200 mL) andbrine (200 mL). The organic layer was dried over MgSO₄ and evaporated toprovide compound D (24.4 g, yield exceeded quantitative) as a yellowishoil. LC-MS [M+H] 717.4 (C₃₅H₅₂N₆O₈S+H, calc: 717.9). Compound D was useddirectly in the next reaction without purification as a dioxanesolution.

Preparation 29: Synthesis of {2-[H-Arg(Pbf)]-aminoethyl}-methyl-carbamicacid benzyl ester (E)

Compound D (24.4 g, ˜30.4 mmol) was dissolved in dioxane (150 mL) andtreated with 4 N HCl/dioxane (150 mL, 600 mmol) at ambient temperaturefor 1 h. The solvent was then evaporated. The residue was suspended ini-PrOH (100 mL) and the mixture was evaporated (procedure was repeatedtwice). The residue was then dried in vacuo to provide compound E (21.1g, yield exceeded quantitative) as a yellowish solid. LC-MS [M+H] 617.5(C₃₀H₄₄N₆O₆S+H, calc: 617.8). Compound E was used directly in the nextreaction without purification as a DMF solution.

Preparation 30: Synthesis of{2-[2-tert-butylmalonyl-Arg(Pbf)]-aminoethyl}-methyl-carbamic acidbenzyl ester (F)

A solution of compound E (21.1 g, ˜30.4 mmol), mono-tert-butyl malonate(5.9 mL, 36.7 mmol), BOP (16.2 g, 36.7 mmol) and DIEA (14.9 mL, 83.5mmol) in DMF (100 mL) was maintained at ambient temperature for 1 h. Thereaction mixture was diluted with EtOAc (1 L) and extracted with water(500 mL), 5% aq. NaHCO₃ (500 mL), water (3×500 mL) and brine (500 mL).The organic layer was dried over MgSO₄, filtered, and then evaporated toprovide compound F (24.5 g, 97%) as a yellowish amorphous solid. LC-MS[M+H] 759.6 (C₃₇H₅₄N₆O₉S+H, calc: 759.9). Compound F was used withoutfurther purification.

Preparation 31: Synthesis ofN-{2-[2-tert-butylmalonyl-Arg(Pfb)]}-N′-methyl-ethane-1,2-diamine (G)

Compound F (12.3 g, 16.7 mmol) was dissolved in methanol (100 mL)followed by the addition of a Pd/C (5% wt, 2.0 g) suspension in water (2mL). The reaction mixture was subjected to hydrogenation (Parrapparatus, 70 psi H₂) at ambient temperature for 1 h. The catalyst wasthen filtered and washed with methanol. The filtrate was evaporated invacuo to provide compound G (10.0 g, 99%) as a colorless amorphoussolid. LC-MS [M+H] 625.5 (C₂₉H₄₈N₆O₇S+H, calc: 625.8). Compound G wasused without further purification.

Preparation 32: Oxycodone Free Base

Oxycodone hydrochloride (10.0 g, 28.5 mmol) was dissolved in chloroform(150 mL) and washed with 5% aq. NaHCO₃ (50 mL). The organic layer wasdried over MgSO₄ and evaporated. The residue was dried in vacuoovernight to provide oxycodone free base (8.3 g, 93%) as a white solid.

Preparation 33: Synthesis ofN—{(S)-4-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-guanidino)-1-[2-(methyl-[(5R,9R,13S,14S)-4,5a-epoxy-6,7-didehydro-14-hydroxy-3-methoxy-17-methylmorphinan-6-oxy]carbonyl-amino)-ethylcarbamoyl]-butyl}-malonamicacid tert-butyl ester (H)

A solution of oxycodone free base (6.6 g, 21.0 mmol) in THF (400 mL) wascooled to −20° C., followed by addition of a 0.5 M solution of KHMDS intoluene (46.3 mL, 23.1 mmol). The obtained solution was then added to asolution of 4-nitro-phenyl chloroformate (4.3 g, 21.0 mmol) in THF (100mL) drop wise over the period of 20 min at ˜20° C. The reaction wasmaintained at −20° C. for an additional 1 h, followed by addition of asolution of compound G (10.0 g, 16.1 mmol) in THF (200 mL) at ˜20° C.The reaction mixture was allowed to warm to ambient temperature andstirred overnight. Solvents were evaporated in vacuo. The resultingresidue was dissolved in EtOAc (20 mL) and precipitated with ether (1L). The formed precipitate was filtrated, washed with ether and dried invacuo to provide compound H (13.6 g, 87%) as an off-white solid. LC-MS[M+H] 966.9 (C₄₈H₆₇N₇O₁₂S+H, calc: 966.2).

Synthesis ofN—{(S)-4-guanidino-1-[2-(methyl-[(5R,9R,13S,14S)-4,5a-epoxy-6,7-didehydro-14-hydroxy-3-methoxy-17-methylmorphinan-6-oxy]carbonyl-amino)-ethylcarbamoyl]-butyl}-malonamicacid (Compound KC-3)

Compound H (13.6 g, 14.1 mmol) was dissolved in a mixture of 5%m-cresol/TFA (100 mL). The reaction mixture was maintained at ambienttemperature for 1 h, followed by dilution with ethyl ether (1 L). Theformed precipitate was filtered, washed with ether and hexane, and driedin vacuo to provide a TFA salt of Compound KC-3 (11.4 g, 81%) as anoff-white solid. LC-MS [M+H] 658.6 (C₃₁H₄₃N₇O₉+H, calc: 658.7).

The TFA salt of crude Compound KC-3 (11.4 g, 11.4 mmol) was dissolved inwater (50 mL). The obtained solution was subjected to HPLC purification.[Nanosyn-Pack YMC-GEL-ODS A (100-10)C-18 column (75×500 mm); flow rate:250 mL/min; injection volume 50 mL; mobile phase A: 100% water, 0.1%TFA; mobile phase B: 100% ACN, 0.1% TFA; isocratic elution at 0% B in 4min, gradient elution from 0% to 10% B in 20 min, isocratic elution at10% B in 30 min, gradient elution from 10% B to 30% B in 41 min;detection at 254 nm]. Fractions containing Compound KC-3 were combinedand concentrated in vacuo. The TFA counterion of the latter was replacedwith an HCl counterion via lyophilization using 0.1N HCl to provide aHCl salt of Compound KC-3 (4.2 g, 41% yield) as a white solid. LC-MS[M+H] 658.6 (C₃₁H₄₃N₇O₉+H, calc: 658.7).

Example 12: Synthesis ofN—((S)-1-{2-[(Dihydrocodein-6-enyloxycarbonyl)-methylamino]-ethylcarbamoyl-4-guanidino}-butyl)-malonamicacid (Compound KC-4)

Preparation 34: Synthesis of tert-butyl 2-(benzylamino)ethylcarbamate(A)

To a solution of tert-butyl 2-aminoethylcarbamate (6.4 g, 40.0 mmol) inmethanol (60 mL) was added benzaldehyde (4.7 g, 44.0 mmol) and molecularsieve 3 Å. After stirring at ambient temperature overnight, the mixturewas cooled down to ca. −10° C. (ice/salt bath) and treated portion wisewith NaBH₄ (9.1 g, 240.0 mmol) over 30 min. After complete addition, thebath was removed and the reaction mixture stirred at ambient temperaturefor 16 h. The solvent was evaporated and the residue taken into EtOAc(150 mL) and poured into water (100 mL). The organic layer was extractedwith 0.5 N HCl (3×100 mL). The combined aqueous solution was cooled to0° C., basified with sat. NaHCO₃ and extracted with CHCl₃ (3×100 mL).The combined organic layers were washed with brine (200 mL). Afterdrying over MgSO₄ and filtering, the solvent was evaporated in vacuo togive compound A (9.2 g, 36.8 mmol, 92%) as a colorless oil. LC-MS [M+H]251.2 (C₁₄H₂₂N₂O₂+H, calc: 251.3). TLC R_(f)(DCM/MeOH 9:1): 0.30.Compound A was used without further purification.

Preparation 35: Synthesis of tert-butyl2-(N-benzyl-N-methylamino)ethylcarbamate (B)

To a cooled (˜5° C.) solution of compound A (6.2 g, 25.0 mmol) and TEA(3.0 g, 29.7 mmol, 4.13 mL) in chloroform (50 mL) was added iodomethane(4.2 g, 29.7 mmol, 1.85 mL). The pressure tube was sealed, and themixture stirred at ambient temperature for 20 h. The mixture was thenprecipitated with ether (300 mL); the white solid was filtered off andwashed with ether (50 mL). The filtrate was concentrated and theresidual yellow oil (5.2 g) was purified by silica gel columnchromatography (2-10% MeOH gradient in DCM) to give compound B (3.3 g,12.5 mmol, 50%) as a colorless oil. TLC R_(f) (DCM/MeOH 9:1): 0.55.LC-MS [M+H] 264.3 (C₁₅H₂₄N₂O₂+H, calc: 264.4).

Preparation 36: Synthesis of tert-butyl 2-(methylamino)ethylcarbamate(C)

To a flask was added 20% Pd(OH)₂ on carbon (3.1 g), compound B (3.3 g,12.5 mmol) in MeOH (200 mL) and water (10 mL), while being exposed to H₂(40 psi). After 2.5 h, the reaction mixture was filtered through celiteand concentrated in vacuo. Water was then added (50 mL) and the mixturebrought to pH 12 (by addition of 1 N NaOH) and extracted with DCM (3×50mL). The combined organic layers were dried over MgSO₄, filtered, andconcentrated in vacuo to give compound C (2.0 g, 11.7 mmol, 94%) as acolorless oil. LC-MS [M+H] 686.5 (C₃₅H₅₁N₅O₇S+H, calc: 685.9). CompoundC was used without further purification.

Preparation 37: Synthesis of[2-(N-dihydrocodein-6-enyloxycarbonyl-N-methylamino)ethyl]carbamic acidtert-butyl ester (D)

To a cooled (−5° C.) solution of hydrocodone (2.9 g, 9.8 mmol, freebase) in anhydrous THF (150 mL) was added drop wise, a 0.5 M solution ofKHMDS in toluene (11.6 mmol, 23.3 mL) over 20 min. The yellow solutionwas stirred at this temperature for 30 min. The solution was addedthrough a cannula to a cooled solution (−30° C.) of 4-nitrophenylchloroformate (1.9 g, 9.5 mmol) in anhydrous THF (40 mL) over 15 min.The bath was removed and the mixture stirred at ambient temperature for15 min until treated drop wise with a solution of compound C (2.3 g,11.6 mmol) in anhydrous THF (15 mL) over 10 min. After stirring atambient temperature for 18 h, the reaction mixture was quenched withsat. NaHCO₃ solution (7 mL). The resulting precipitate was filtered,washed with EtOAc (30 mL) and the filtrate concentrated in vacuo. Theresidue was taken into EtOAc (300 mL) and washed with a mixture of water(100 mL) and 2% aq. H₂SO₄ (30 mL). The aqueous layer was basified with 2N NaOH to pH 12 and extracted with EtOAc (2×200 mL). The combinedorganic layers were washed with water (2×400 mL) and brine (300 mL),dried over MgSO₄, filtered and concentrated in vacuo to give a yellowishfoamy solid (5.9 g), which was purified by HPLC. [Nanosyn-Pack Microsorb(100-10)C-18 column (50×300 mm); flow rate: 100 mL/min; injectionvolume: 65 mL; mobile phase A: 100% water, 0.1% TFA; mobile phase B:100% acetonitrile, 0.1% TFA; isocratic elution at 10% B in 5 min,gradient elution to 18% B in 8 min, isocratic elution at 18% B in 20min, gradient elution from 18% B to 40% B in 44 min; detection at UV 254nm]. Fractions containing the desired compound were combined andconcentrated in vacuo. Traces of water were removed by treating theresidue with toluene (30 mL) followed by evaporation in vacuo (procedurewas repeated twice). The isolated fractions are a 1:1 mixture ofcompound D and the boc deprotected compound E (4.37 g, 7.85 mmol, 83%).LC-MS [M+H] 500.2 (C₂₇H₃₇N₃O₆+H, calc: 500.6). Retention time[Chromolith SpeedRod RP-18e C18 column (4.6×50 mm); flow rate: 1.5ml/min; mobile phase A: 0.1% TFA/water; mobile phase B 0.1% TFA/ACN;gradient elution from 5% B to 100% B over 9.6 min, detection 254 nm]:3.52 min (compound D), 1.82 (compound E).

Preparation 38: Synthesis ofdihydrocodein-6-enyl-2-aminoethylmethylcarbamate (E)

A solution of compound D (4.4 g, 8.8 mmol) in DCM (40 mL) was treatedwith 4 M HCl in dioxane (105 mmol, 26 mL), leading to some precipitateformation. The mixture was homogenized by addition of acetonitrile (20mL) and stirred at ambient temperature for 45 min. Ether (400 mL) wasadded and the resulting white precipitate filtered, washed with ether(50 mL) and hexane (50 mL) and then dried in vacuo to give compound E asan off-white solid (2.4 g, 4.7 mmol, 58%). LC-MS [M+H] 400.3(C₂₂H₂₉N₃O₄+H, calc: 400.5). Compound E was used without furtherpurification.

Preparation 39: Synthesis of {2-[boc-Arg(Pbf)]-aminomethyl}-ethylcarbamic acid hydrocodone ester (F)

Compound E (2.0 g, 4.0 mmol), Boc-Arg(Pbf)-OH (2.0 g, 3.8 mmol) and HATU(1.7 g, 4.3 mmol) were dissolved in DMF (40 mL), brought to ˜5° C. andtreated drop wise with DIPEA (3.2 mL, 18.1 mmol) over 10 min. Thereaction mixture was stirred at ˜5° C. for an additional 10 min and thenwarmed to ambient temperature, followed by stirring for 30 min. Thereaction was then diluted with EtOAc (200 mL) and poured into water (250mL). The layers were separated, the aqueous extracted with EtOAc (2×150mL) and the combined organic layers washed with 2% aq. H₂SO₄ (30 mL),water (2×250 mL) and brine (250 mL). The organic layer was dried overMgSO₄, filtered and concentrated in vacuo to give compound F (3.0 g, 3.2mmol, 83%) as a yellowish foamy solid. LC-MS [M+H] 908.7(C₄₆H₆₅N₇O₁₀S+H, calc: 909.1). Compound F was used without furtherpurification.

Preparation 40: Synthesis of {2-[H-Arg(Pbf)]-aminomethyl}-ethyl carbamicacid hydrocodone ester (G)

A solution of compound F (3.0 g, 3.3 mmol) in DCM (20 mL) was treatedwith 4 M HCl in dioxane (39 mmol, 9.8 mL) and stirred at ambienttemperature for 30 min. Ether (500 mL) was added and the resulting whiteprecipitate was filtered, washed with ether (50 mL) and hexane (50 mL)and then dried in vacuo to give compound G as an off-white solid (2.7 g,3.0 mmol, 93%). LC-MS [M+H] 808.7 (C₄₁H₅₇N₇O₈S+H, calc: 809.0). CompoundG was used without further purification.

Preparation 41: Synthesis ofN—((S)-1-{2-[(Dihydrocodein-6-enyloxycarbonyl)-methylamino]-ethylcarbamoyl-4-guanidino(Pbf))-butyl}-malonamicacid tert-butyl ester (H)

To a cooled solution (˜5° C.) of compound G (2.7 g, 3.0 mmol) was addedmono tert-Butyl malonate (474 mg, 3.0 mmol, 438 μL) in DMF (25 mL)followed by BOP (1.4 g, 3.2 mmol) over 5 min and finally by DIEA (1.6 g,12.1 mmol, 2.1 mL) drop wise over 10 min. After an additional 15 min,the ice bath was removed and the mixture stirred at ambient temperature.After 45 min, the reaction mixture was diluted with EtOAc (300 mL) andpoured into water (200 mL). The layers were separated and the aqueouslayer extracted with EtOAc (2×250 mL). The combined organic layers werewashed with water (500 mL), 2% aq. H₂SO₄ (100 mL), water (3×500 mL) andbrine (2×500 mL). After drying over MgSO₄, the solvent was evaporated invacuo and the residue dried under high vacuum to give H (1.7 g, 1.8mmol, 58%) as a yellowish solid. LC-MS [M+H] 950.8 (C₄₈H₆₇N₇O₁₁S+H,calc: 951.2). Compound H was used without further purification.

Synthesis ofN—((S)-1-{2-[(Dihydrocodein-6-enyloxycarbonyl)-methylamino]-ethylcarbamoyl-4-guanidino-butyl)-malonamicacid (Compound KC-4)

A solution of compound H (1.7 g, 1.8 mmol) in 5% m-cresol/TFA (45 mL)was stirred at ambient temperature. After 1 h, the mixture was dilutedwith ether (300 mL). The resulting fine suspension was filtered, thesolid washed with ether (30 mL) and hexane (30 mL) and dried in vacuofor 15 min. The crude material was dissolved in water (35 mL) andpurified by HPLC [Nanosyn-Pack Microsorb (100-10)C-18 column (50×300mm); flow rate: 100 mL/min; injection volume: 35 mL; mobile phase A:100% water, 0.1% TFA; mobile phase B: 100% acetonitrile, 0.1% TFA;gradient elution 0 to 10% B in 10 min, isocratic elution at 10% B in 20min, gradient elution from 10% B to 42% B in 60 min; detection at UV 254nm]. Fractions containing the desired compound were combined andconcentrated in vacuo. The residue was treated with toluene (50 mL) toremove traces of water and co-evaporated in vacuo (procedure repeatedtwice). The residue was dissolved in acetonitrile (5 mL), treated with2.0 M HCl in ether (20 mL), followed by dilution with ether (100 mL).The resulting solid was filtered, washed with ether (20 mL) and hexane(20 mL) and dried in vacuo overnight to provide Compound KC-4 (1.1 g,86% yield) as a white solid, hydrochloride salt. LC-MS [M+H] 642.5(C₃₁H₄₃N₇O₈+H, calc: 642.7). Purity >95% (UV/254 nm). Retention time[Chromolith SpeedRod RP-18e C18 column (4.6×50 mm); flow rate: 1.5ml/min; mobile phase A: 0.1% TFA/water; mobile phase B 0.1% TFA/ACN;gradient elution from 5% B to 100% B over 9.6 min, detection 254 nm]:2.24 min.

Example 13: Synthesis of6-{[2-(2-Acetylamino-5-guanidino-pentanoylamino)-ethyl]-[(5R,9R,13S,14S)-4,5a-epoxy-6,7-didehydro-14-hydroxy-3-methoxy-17-methylmorphinan-6-oxy]-1-enyloxycarbonyl-amino}-hexanoicacid (Compound KC-5)

Preparation 42: Synthesis of6-[Benzyloxycarbonyl-(2-tert-Butoxycarbonylamino-ethyl)-amino]-hexanoicacid ethyl ester (B)

Compound A (26.8 g, 88.6 mmol) was dissolved in DCM (200 mL) at ambienttemperature. NEt₃ (12.5 mL, 88.6 mmol) was added, followed by Cbz-C1(Z-C1) (12.5 mL, 88.6 mmol). The reaction mixture was stirred at ambienttemperature under N₂ for 2 h. The reaction mixture was treated withNaHCO₃ (30 mL, aq. sat.). The layers were separated and the organiclayer was dried over MgSO₄, filtered and concentrated. The residue waspurified by silica gel chromatography, using 4/1 hexanes/EtOAc, to givecompound B as a colorless oil (22.5 g, 66.5 mmol, 75%).

Preparation 43: Synthesis of Intermediate (C)

Compound B (22.0 g, 50.4 mmol) was dissolved in DCE (100 mL) at ambienttemperature. NEt₃ (8.5 mL, 61 mmol) was added, followed by (Boc)₂O (33.0g, 151.2 mmol) and DMAP (615 mg, 5.0 mmol). The reaction mixture wasstirred at ambient temperature under N₂ for 2 h and then heated at 60°C. for 16 h. The reaction mixture was concentrated, and the residue waspurified by silica gel chromatography, using 4/1 hexanes/EtOAc, to givecompound C as a colorless oil (23.2 g, 41.9 mmol, 86%). MS: (m/z) calc:536.6, observed (M+Na⁺) 560.1.

Preparation 44: Synthesis of Intermediate (D)

Compound C (22.5 g, 41.9 mmol) was dissolved in EtOH (50 mL). Themixture was degassed and saturated with N₂. Pd/C (500 mg, 5% on carbon)was added. The mixture was shaken in a Parr hydrogenator flask under 2atm H₂ for 2 h. The mixture was then filtered through a celite pad andthe filtrate was concentrated to give crude compound D as a colorlessoil (21.0 g, 52.2 mmol, crude). This material was used without furtherpurification.

Preparation 45: Synthesis of Intermediate (E)

Compound D (21.0 g, 52.2 mmol, crude) and NEt₃ (11.0 mL, 78.3 mmol) weremixed together with DCM (150 mL). The mixture was added to a pre-chilled(ice/water bath) phosgene solution in toluene (41.2 mL, 20% wt intoluene, ˜83.3 mmol). The reaction mixture was stirred at 0° C. for 2 h.It was then concentrated to one-third of its original volume and dilutedwith ether (50 mL). The mixture was filtered through filter paper. Thefiltrate was concentrated to give compound E as a white solid (20.0 g,43.1 mmol, 82%) MS: (m/z) calc: 464.2, observed (M+Na⁺) 487.7. CompoundE was used without further purification.

Preparation 46: Synthesis of Intermediate (F)

Oxycodone free base (1.0 g, 3.2 mmol) was dissolved in dry THF(degassed) (15 mL) and the mixture was cooled to ˜10° C. using a dryice/acetone bath. KHMDS (7.6 mL, 3.8 mmol, 0.5 M in toluene) was addedvia syringe. The mixture was stirred under N₂ at a temperature below −5°C. for 30 min. Compound E (1.5 g, 3.2 mmol) in THF (10 mL) was thenadded via syringe over 5 min. The mixture was stirred at ˜5° C. for 30min. The reaction was continued at ambient temperature for 2 h. NaHCO₃(10 mL, sat. aq.) was added. The mixture was concentrated in vacuo tohalf of its initial volume. EtOAc (20 mL) was added and the layers wereseparated. The organic phase was further washed with water (20 mL) andbrine (20 mL), followed by concentration with the resulting residuepurified by silica gel chromatography (DCM/MeOH (gradient 100/1 to100/15)) to afford a colorless oil (˜1.7 g, 3.1 mmol, 97%). Thismaterial was dissolved in a mixture of DCM/TFA (5 mL/5 mL) at ambienttemperature and stirred for 1 h. It was then concentrated in vacuo toafford compound F as its TFA salt (1.8 g, 2.7 mmol, 88%). MS: (m/z)calc: 543.7, observed (M+H⁺) 545.2. Compound F was used without furtherpurification.

Preparation 47: Synthesis of Intermediate (G)

Compound F (1.8 g, 2.6 mmol) was dissolved in DMF (20 mL) with stirring.Boc-Arg(Pbf)-OH (1.4 g, 2.7 mmol), HATU (1.1 g, 2.9 mmol) and DIPEA (1.4mL, 8.0 mmol) were added with stirring. The reaction was continued atambient temperature for 2 h. The mixture was then concentrated, and theresidue was partitioned between EtOAc and water (30 mL/20 mL). Theorganic layer was separated, washed with water (20 mL), brine (20 mL),dried over Na₂SO₄ and concentrated to afford crude compound G (1.5 g,1.4 mmol, 54%). MS: (m/z) calc: 1052.3, observed (M+H⁺) 1053.9. CompoundG was used without further purification.

Preparation 48: Synthesis of Intermediate (H)

Crude compound G (1.5 g, 1.4 mmol)) was taken into dioxane (3 mL) andcooled in an ice/water bath. An HCl solution in dioxane (4 N, 10 mL, 40mmol) was added and the mixture was stirred at ambient temperature for 3h and then concentrated in vacuo to afford a white foam. This materialwas dissolved in a mixture of DIPEA (0.8 mL 4.3 mmol) in DCM (20 mL).Acetic anhydride (0.2 mL, 2.1 mmol) was added. The reaction mixture wasstirred at ambient temperature for 2 h. NaHCO₃ (20 mL, sat. aq.) wasadded. The layers were separated and the DCM layer was dried overNa₂SO₄, filtered and concentrated to afford intermediate compound H(0.85 g, crude). Compound H was used without further purification.

Synthesis of6-{[2-(2-Acetylamino-5-guanidino-pentanoylamino)-ethyl]-[(5R,9R,13S,14S)-4,5a-epoxy-6,7-didehydro-14-hydroxy-3-methoxy-17-methylmorphinan-6-oxy]-1-enyloxycarbonyl-amino}-hexanoicacid (Compound KC-5)

Compound H (0.85 g, crude) was dissolved in a mixture of m-Cresol (0.5mL) in TFA (20 mL). The mixture was stirred at ambient temperature for 2h. The mixture was concentrated in vacuo. The residue was taken intoMeOH (3 mL) and added drop wise to a stirred HCl solution in ether (20mL, 2 M, 40 mmol). The resulting white solid (compound I) was filteredand washed with ether (3×10 mL). Compound I was then dissolved in amixture of THF/H₂O (2 mL/2 mL) at ambient temperature. LiOH (41 mg, 1.7mmol) was added in one portion. The mixture was stirred for 4 h. Themixture was then acidified by adding AcOH until pH ˜6. The mixture wasthen concentrated and the residue was purified by prep HPLC, usingRP-18e C18 column (4.6×50 mm); flow rate: 1.5 ml/min; mobile phase A:0.1% TFA/water; mobile phase B 0.1% TFA/CH₃CN; gradient elution.Lyophilization of the collected fractions afforded Compound KC-5 (TFAsalt) as a white solid. The solid was treated with 0.1 N HCl (aq.) andlyophilized to give the corresponding HCl salt of Compound KC-5 as awhite foam (406 mg, 38% from compound E, 100% purity). MS: (m/z) calc:713.8, observed (M+H⁺) 714.5.

Example 14:({(S)-2-((S)-2-Acetylamino-5-guanidino-pentanoylamino)-3-[(oxycodone-enyloxycarbonyl)-methyl-amino]-propionyl}-methyl-amino)-aceticacid (Compound KC-6)

Preparation 49:(S)-2-tert-Butoxycarbonylamino-3-(2-nitro-benzenesulfonylamino)-propionicacid (A)

(S)-2-tert-Butoxycarbonylamino-3-amino-propionic acid (14.9 g, 73.2mmol) was dissolved in a mixture of THF (45 mL) and 3 N aq. NaOH (45mL). The reaction mixture was cooled to ˜10° C. and nosyl chloride (17.9g, 80.5 mmol) was added as a THF solution (75 mL) drop wise over 30 min.The reaction mixture was stirred at ˜10° C. for 45 min followed bystirring at ambient temperature for 30 min. The reaction mixture wasdiluted with water (150 mL), acidified with 2% aqueous H₂SO₄ (to pH ˜2)and diluted with additional water (450 mL). The product was extractedwith EtOAc (600 mL total) and washed with water (3×400 mL) and brine(100 mL). The organic layer was separated, dried over Na₂SO₄, filteredand condensed in vacuo to afford compound A (20.0 g, 70% yield) as acream solid. LC-MS [M+H-Boc] 290.3 (C₁₄H₁₉N₃O₈S+H, calc: 390.4).Purity >95% (UV/254 nm). Compound A was used without furtherpurification.

Preparation 50:{[(S)-2-tert-Butoxycarbonylamino-3-(2-nitro-benzenesulfonylamino)-propionyl]-methyl-amino}-aceticacid ethyl ester (B)

Free basing procedure of Sarcosine ethyl ester: Sarcosine ethyl esterhydrochloride (39.3 g, 256.8 mmol) was dissolved in water (300 mL),washed with Et₂O (2×100 mL), pH adjusted to ˜pH 8, extracted with CHCl₃(3×100 mL) and dried over Na₂SO₄ and finally filtered.

To a solution of compound A (10.0 g, 25.7 mmol) in DMF (100 mL) wasadded HOBt (5.2 g, 38.5 mmol) and the reaction mixture was cooled to˜10° C. To this reaction mixture, EDC-HCl (5.4 g, 28.2 mmol) was addedin portions over 10 min and stirred at ˜10° C. for 20 min. To thereaction mixture, Sarcosine ethyl ester (256.8 mmol) in CHCl₃ (300 mL)was added drop wise over 30 min. The reaction mixture was stirred atthis temperature for 30 min followed by stirring at ambient temperatureovernight. Solvents were then removed in vacuo, and the residue wasdissolved in EtOAc (500 mL), washed with water (3×300 mL), saturatedaqueous NaHCO₃ (2×300 mL) and brine (100 mL). The organic layer wasseparated, dried over Na₂SO₄ and concentrated in vacuo to affordcompound B (11.5 g, 91%) as a cream solid. LC-MS [M+H]489.5(C₁₉H₂₈N₄O₉S+H, calc: 489.3). Purity >95% (UV/254 nm). Compound B wasused without further purification.

Preparation 51:({(S)-2-tert-Butoxycarbonylamino-3-[methyl-(2-nitro-benzenesulfonyl)-amino]-propionyl}-methyl-amino)-aceticacid ethyl ester (C)

Compound B (8.0 g, 16.3 mmol) was dissolved in DMF (40 mL) and thereaction mixture was cooled to ˜10° C. To the reaction mixture was addedK₂CO₃ (6.8 g, 49.1 mmol) followed by addition of MeI (5.1 mL, 81.9 mmol)drop wise and stirred at 0° C. for 1 h. The reaction mixture wasfiltered and washed with EtOAc. Solvents removed in vacuo and theresidue was dissolved in EtOAc (250 mL) and poured into water (500 mL),extracted with EtOAc (2×250 mL), and washed with water (250 mL) andbrine (100 mL). The organic layer was dried over Na₂SO₄, filtered, andthen concentrated in vacuo, to afford compound C (8.1 g, 98% yield) as acream solid. LC-MS [M+H] 503.1 (C₂₀H₃₀N₄O₉S+H, calc: 503.5). Purity >95%(UV/254 nm). Compound C was used without further purification.

Preparation 52:({(S)-2-Amino-3-[methyl-(2-nitro-benzenesulfonyl)-amino]-propionyl}-methyl-amino)-aceticacid ethyl ester (D)

Compound C (6.9 g, 13.8 mmol) was dissolved in DCM (45 mL) and thentreated with 4 M HCl in dioxane (40 mL) at ambient temperature. Thereaction mixture was stirred at ambient temperature for 90 min. Themixture was concentrated in vacuo to a total volume of ˜25 mL, and Et₂O(400 mL) was added. The precipitated product was filtered off, washedwith Et₂O (250 mL), and hexane (250 mL) and finally dried in vacuo toafford compound D (6.3 g, 100% yield) as a cream solid. LC-MS [M+H]403.3 (C₁₅H₂₂N₄O₇S+H, calc: 403.4). Purity >95% (UV/254 nm). Compound Dwas used without further purification.

Preparation 53:({(S)-2-[(S)-5-({Amino-[(Z)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-2-tert-butoxycarbonylamino-pentanoylamino]-3-[methyl-(2-nitro-benzenesulfonyl)-amino]-propionyl}-methyl-amino)-aceticacid ethyl ester (E)

To a solution of Boc-Arg(Pbf)-OH (7.3 g, 13.8 mmol), DIPEA (7.7 mL, 44.2mmol) in DMF (35 mL) was added HATU (5.8 g, 15.2 mmol) and stirred at 5°C. for 15 min. To this reaction mixture, compound D (6.3 g, 13.8 mmol)was added and stirred at ambient temperature for 1 h. DMF was thenremoved in vacuo to a total volume of ˜15 mL. The reaction mixture wasdiluted with EtOAc (250 mL) and poured into water (500 mL), extractedwith EtOAc (2×250 mL), and washed with 2% aqueous H₂SO₄ (150 mL), water(150 mL) and brine (150 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered and then evaporated to give an oily residue, which wasdried overnight under high vacuum to give compound E (7.4 g, 59%) as anoff-white solid. LC-MS [M+H] 911.5 (C₃₉H₅₈N₈O₁₃S+H, calc: 912.05).Purity >95% (UV/254 nm). Compound E was used without furtherpurification.

Preparation 54:({(S)-2-[(S)-2-Amino-5-({amino-[(Z)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-pentanoylamino]-3-[methyl-(2-nitro-benzenesulfonyl)-amino]-propionyl}-methyl-amino)-aceticacid ethyl ester (F)

Compound E (7.4 g, 8.2 mmol) in DCM (24 mL) was treated with 4 M HCl indioxane (24 mL) at ambient temperature. The reaction mixture was stirredat ambient temperature for 1 h. DCM and most of the dioxane were removedin vacuo to a total volume of −15 mL, and Et₂O (300 mL) was added.Precipitated product was filtered off, washed with Et₂O (150 mL) andhexane and finally dried in vacuo to afford compound F (6.34 g, 100%yield) as a cream solid. LC-MS [M+H] 811.4 (C₃₄H₅₀N₈O₁₁S₂+H, calc:811.94). Purity >95% (UV/254 nm). Compound F was used without furtherpurification.

Preparation 55:({(S)-2-[(S)-2-Acetylamino-5-({amino-[(Z)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-pentanoylamino]-3-[methyl-(2-nitro-benzenesulfonyl)-amino]-propionyl}-methyl-amino)-aceticacid ethyl ester (G)

To a solution of compound F (6.6 g, 7.8 mmol) in CHCl₃ (50 mL) at 5° C.was added DIPEA (4.8 mL, 27.4 mmol) followed by Ac₂O (0.9 mL, 9.4 mmol).The reaction mixture was stirred at ambient temperature for 30 min.Solvents were removed in vacuo, and then the residue was diluted withwater (500 mL) and EtOAc (500 mL). The organic layer was separated andwashed with water (300 mL), 2% aqueous H₂SO₄ (200 mL), water (2×300 mL)and brine (100 mL). The organic layer was separated, dried over Na₂SO₄and solvent removed in vacuo to afford compound G (5.5 g, 82%). LC-MS[M+H] 853.4 (C₃₆H₅₂N₈O₁₂S₂+H, calc: 853.9). Purity >95% (UV/254 nm).Compound G was used without further purification.

Preparation 56:({(S)-2-[(S)-2-Acetylamino-5-({amino-[(Z)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-pentanoylamino]-3-methylamino-propionyl}-methyl-amino)-aceticacid ethyl ester (H)

To a solution of compound G (5.5 g, 6.5 mmol) in DMF (21 mL) at ambienttemperature was added K₂CO₃ (8.9 g, 64.5 mmol) followed by thioglycerol(5.6 mL, 64.5 mmol). The reaction mixture was stirred at ambienttemperature for 1 h, filtered off and DMF was removed in vacuo. Theresidue was diluted with water (500 mL) and extracted with EtOAc (2×300mL) and CHCl₃ (2×300 mL). Combined organic layers were dried and removalof the solvents in vacuo afforded the crude product. The crude productwas purified by flash chromatography eluting with EtOAc followed by 10%MeOH in CHCl₃ to afford compound H (1.3 g, 30%). LC-MS [M+H] 668.3(C₃₀H₄₉N₇O₈S+H, calc: 667.8). Purity >95% (UV/254 nm).

Preparation 57:({(S)-2-[(S)-2-Acetylamino-5-(amino-[(Z)-2,2,4,6,7-pentamethyl-2,3-dihydro-benzofuran-5-sulfonylimino]-methyl}-amino)-pentanoylamino]-3-[(oxycodone-enyloxycarbonyl)-methyl-amino]-propionyl}-methyl-amino)-aceticacid ethyl ester (I)

To a solution of oxycodone free base (2.0 g, 6.3 mmol) in THF (100 mL)at ˜60° C. was added 0.5 M KHMDS (13.9 mL, 7.0 mmol) drop wise. Thereaction mixture was stirred for 30 min and then transferred to asolution of 4-nitrophenyl chloroformate (1.3 g) in THF (100 mL) at −60°C. and stirred for 30 min. A solution of amine compound H (3.2 g, 4.9mmol) was added as a THF (20 mL) solution to the reaction mixture. Afterstirring at ˜60° C. for 15 min, the cooling bath was removed and thereaction was stirred at ambient temperature overnight. Another portion(1.0 g, 3.2 mmol) of oxycodone free base was activated using the aboveprocedure and added to the reaction mixture as above, and stirringcontinued overnight. The reaction was determined to be complete byLC-MS. The solvents were removed, and the residue was dissolved in MeOH(˜25 mL) and precipitated with Et₂O (400 mL). The precipitate was washedwith Et₂O and hexane and dried in vacuo. The product was dissolved inwater and DMSO and purified by HPLC. [Nanosyn-Pack Microsorb(100-10)C-18 column (50×300 mm); flow rate: 100 mL/min; injection volume15 mL; mobile phase A: 100% water, 0.1% TFA; mobile phase B: 100% ACN,0.1% TFA; gradient elution from 0% to 33% B in 33 min, isocratic elutionat 33% B in 30 min, gradient elution from 33% B to 50% B in 33 min;detection at 254 nm]. Desired fractions were combined and dried in vacuoto afford compound I (5 g, 92% yield)). LC-MS [M+H] 979.6(C₄₈H₆₆N₈O₁₂S+H, calc: 980.15). Purity >95% (UV/254 nm).

Preparation 58:({(S)-2-((S)-2-Acetylamino-5-guanidino-pentanoylamino)-3-[(oxycodone-enyloxycarbonyl)-methyl-amino]-propionyl}-methyl-amino)-aceticacid ethyl ester (J)

Compound I (5 g, 4.5 mmol) was treated with 5% m-cresol in TFA (25 mL).After 1 h, ether (400 mL) was added to the reaction mixture. Theprecipitated product was filtered off, washed with Et₂O and hexane anddried in vacuo to afford compound J (3.2 g, 65% yield). LC-MS [M+H]757.7 (C₃₆H₅₂N₈O₁₀+H, calc: 757.9). Purity >95% (UV/254 nm). Compound Jwas used without further purification.

({(S)-2-((S)-2-Acetylamino-5-guanidino-pentanoylamino)-3-[(oxycodone-enyloxycarbonyl)-methyl-amino]-propionyl}-methyl-amino)-aceticacid (Compound KC-6)

Compound J was treated with 2 N aq. HCl (75 mL) and heated at 55° C. for6.5 h. Heating was removed and the reaction mixture was cooled to ˜5° C.and pH was adjusted to ˜pH 6 with aqueous saturated NaHCO₃. Most of thewater was removed in vacuo to a total volume of ˜50 mL. This solutionwas subjected to HPLC purification. [Nanosyn-Pack Microsorb (100-10)C-18column (50×300 mm); flow rate: 100 mL/min; injection volume 15 mL;mobile phase A: 100% water, 0.1% TFA; mobile phase B: 100% ACN, 0.1%TFA; isocratic elution at 0% B in 2 min, gradient elution from 0% to 8%B in 14 min, isocratic elution at 8% B in 30 min, gradient elution from8% B to 33% B in 55 min; detection at 254 nm]. Desired fractions werecombined and dried in vacuo, followed by lyophilization using 0.1 N HClto afford Compound KC-6 as a HCl salt (1.5 g, 48% yield). LC-MS [M+H]729.6 (C₃₄H₄₈N₈O₁₀+H, calc: 729.8). Purity >95% (UV/254 nm).

Biological Data Example 15: Pharmacokinetics of Oxycodone ProdrugFollowing PO Administration to Rats

This Example compares the plasma concentrations of oxycodone in ratsfollowing oral (PO) administration of oxycodone6-(N-methyl-N-(2-N′-acetylarginylamino)) ethylcarbamate (produced asdescribed in Example 10 and herein also referred to as Compound KC-2) oroxycodone.

Compound KC-2 and oxycodone were each dissolved in saline and dosed atequimolar doses (20 mg/kg and 10 mg/kg, respectively) via oral gavageinto jugular vein-cannulated male Sprague Dawley rats; four rats weredosed per group. At specified time points, blood samples were drawn,harvested for plasma via centrifugation at 5,400 rpm at 4° C. for 5 min,and 100 microliters (μl) plasma transferred from each sample into afresh tube containing 1 μl of formic acid. The tubes were vortexed for5-10 seconds, immediately placed in dry ice and then stored untilanalysis by high performance liquid chromatography/mass spectrometry(HPLC/MS).

Table 1 indicates plasma C_(max) (maximum plasma concentration) andT_(max) (time after administration when the maximum plasma concentrationwas reached) values of oxycodone (average±standard deviation) for eachgroup of 4 rats. Also indicated are the C_(max) and T_(max) values foroxymorphone, a metabolite of oxycodone.

TABLE 1 Plasma C_(max) and T_(max) values of oxycodone (OC) andoxymorphone (OM) in rats dosed PO with oxycodone or Compound KC-2C_(max) OC Compound (ng/mL T_(max) OC C_(max) OM T_(max) OM administeredOC) (hr) (ng/mL OM) (hr) Oxycodone 14.7 ± 6.5  0.63 ± 0.43 18.4 ± 10.00.50 ± 0.35 Compound 3.8 ± 1.1 3.8 ± 1.5 3.9 ± 1.6 3.8 ± 1.5 KC-2

FIG. 4 compares mean plasma concentrations (±standard deviations) overtime of oxycodone following PO administration of 20 mg/kg Compound KC-2(solid line) or 10 mg/kg oxycodone (dashed line) to rats.

The results in Table 1 and FIG. 4 indicate that administration ofCompound KC-2 yields oxycodone plasma concentrations that exhibit asuppressed C_(max) and delayed T_(max) compared to administration ofoxycodone.

Example 16: Pharmacokinetics of Oxycodone Prodrug Following IVAdministration to Rats

This Example compares the plasma concentrations of prodrug and oxycodonein rats following intravenous (IV) administration of oxycodone6-(N-methyl-N-(2-N′-acetylarginylamino)) ethylcarbamate (produced asdescribed in Example 10 and herein also referred to as Compound KC-2).

Compound KC-2 was dissolved in saline and injected into the tail vein of4 jugular vein-cannulated male Sprague Dawley rats at a dose of 2 mg/kg.At specified time points, blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 microliters(μl) plasma transferred from each sample into a fresh tube containing 1μl of formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored until analysis by high performanceliquid chromatography/mass spectrometry (HPLC/MS).

Table 2 indicates plasma C_(max) values (average±standard deviation) ofCompound KC-2, oxycodone and oxymorphone (a metabolite of oxycodone).

TABLE 2 Plasma C_(max) values of Compound KC-2, oxycodone andoxymorphone in rats dosed IV with Compound KC-2 Compound in plasmameasured Cmax (ng/mL) Compound KC-2  2680 ± 755 Oxycodone 0.798 ± 0.1Oxymorphone 0.118 ± 0.1

FIG. 5 compares mean plasma concentrations (±standard deviations) overtime of Compound KC-2 (solid line) and oxycodone (dashed line) followingIV administration of 2 mg/kg Compound KC-2 to rats. Numbers on theY-axis also depict the Cmax values of Compound KC-2 and oxycodone,respectively.

Table 2 and FIG. 5 demonstrate that the plasma concentration ofoxycodone in rats administered Compound KC-2 IV is only 0.03% of theplasma concentration of Compound KC-2, indicating that IV administrationof Compound KC-2 does not lead to significant release of oxycodone.

Example 17: In Vitro Stability of Oxycodone Prodrug

This Example demonstrates the stability of oxycodone6-(N-methyl-N-(2-N′-acetylarginylamino)) ethylcarbamate (produced asdescribed in Example 10 and herein also referred to as Compound KC-2) toa variety of readily available household chemicals and enzymepreparations.

Compound KC-2 was exposed at room temperature (RT) or 80° C. for either1 or 24 hours (hr) to the following household chemicals: vodka (40%alcohol), baking soda (saturated sodium bicarbonate solution, pH 9),WINDEX® with Ammonia-D (pH11) and vinegar (5% acetic acid). CompoundKC-2 was also exposed to the following enzyme-containing compositions atRT for 1 or 24 hr: GNC® Super Digestive (2 capsules of GNC SuperDigestive Enzymes dissolved in 5 mL of water), tenderizer (Adolf's meattenderizer, primarily papain, dissolved in water to a concentration of0.123 g/mL to approximate the concentration of a marinade given on thebottle label), and subtilisn (8 tablets of ULTRAZYME® contact lenscleaner (Advanced Medical Optics) dissolved in 4 mL water). Samples wereincubated as described and aliquots removed at 1 hr and 24 hr andstabilized by adding each to a solution of 50% or 100% of 85% phosphoricacid solution to achieve a final pH of less than or equal to pH 4. Thestabilized aliquots were then diluted 4- to 6-fold with water,vortex-mixed and applied to HPLC.

FIG. 6 demonstrates the release of oxycodone when Compound KC-2 wasexposed to the various household chemicals and enzyme-containingcompositions described above. The percentage of Compound KC-2 remainingafter exposure is indicated by the solid black bars and percentageconversion of Compound KC-2 to oxycodone is indicated by the lightlyshaded bars with a black outline. These results indicate that exposureof Compound KC-2 to these various conditions leads to substantially lessthan 10% conversion to oxycodone.

Example 18: In Vitro IC50 Data of Several Candidate Trypsin Inhibitors

Several candidate trypsin inhibitors, namely Compounds 101-105, 107 and108 were produced as described in the Examples herein. Compound 106(also known as 4-aminobenzamidine), Compound 109 and Compound 110 areavailable from Sigma-Aldrich (St. Louis, Mo.).

The half maximal inhibitory concentration (IC50 or IC₅₀) values of eachof Compounds 101-110 as well as of SBTI and BBSI were determined using amodified trypsin assay as described by Bergmeyer, H U et al, 1974,Methods of Enzymatic Analysis Volume 1, 2^(nd) edition, 515-516,Bergmeyer, HU, ed., Academic Press, Inc. New York, N.Y.

Table 3 indicates the IC50 values for each of the designated trypsininhibitors.

TABLE 3 IC50 values of certain trypsin inhibitors Compound IC50 value101 2.0E−5 102 7.5E−5 103 2.3E−5 104 2.7E−5 105 4.1E−5 106 2.4E−5 1071.9E−6 108 8.8E−7 109 9.1E−7 110 1.8E−5 SBTI 2.7E−7 BBSI 3.8E−7

The results of Table 3 indicate that each of Compounds 101-110 exhibitstrypsin inhibition activity.

Example 19: Effect of Trypsin Inhibition on In Vitro Trypsin-MediatedTrypsin Release of Oxycodone from Compound KC-2

Compound KC-2 (which can be prepared as described in Example 10) wasincubated with trypsin from bovine pancreas (Catalog No. T8003, Type I,˜10,000 BAEE units/mg protein, Sigma-Aldrich), in the absence orpresence of Compound 109 (Catalog No. N0289, Sigma-Aldrich). WhenCompound 109 was part of the incubation mixture, Compound KC-2 was added5 min after the other incubation components. Specifically, the reactionsincluded 0.523 mg/mL (0.761 mM) Compound KC-2.2HCl, 0.0228 mg/mLtrypsin, 22.5 mM calcium chloride, 172 mM Tris pH 8 and 0.00108 mg/mL (2μM) Compound 109 or 0.25% DMSO depending on whether inhibitor wasincluded in the incubation. The reactions were conducted at 37° C. for24 hr. Samples were collected at specified time points, transferred into0.5% formic acid in acetonitrile to stop trypsin activity and stored atless than −70° C. until analysis by LC-MS/MS.

FIG. 7 indicates the results of exposure of Compound KC-2 to trypsin inthe absence of any trypsin inhibitor (solid symbols) or in the presenceof Compound 109 (open symbols). The square symbols indicate thedisappearance of Compound KC-2, and the triangle symbols depict theappearance of oxycodone, over time under the conditions described inthis Example.

The results in FIG. 7 indicate that a trypsin inhibitor of theembodiments can attenuate trypsin-mediated release of oxycodone fromCompound KC-2. In addition, such a trypsin inhibitor can thwart theability of a user to apply trypsin to effect the release of oxycodonefrom Compound KC-2.

Table 4 indicates the results of exposure of Compound KC-2 to trypsin inthe absence and presence of Compound 109. The results are expressed ashalf-life of prodrug when exposed to trypsin (i.e., Prodrug trypsinhalf-life) in hours and rate of formation of oxycodone per unit totrypsin.

TABLE 4 In Vitro Trypsin Conversion of Compound KC-2 to Oxycodone Notrypsin inhibitor With trypsin inhibitor Rate of Rate of oxycodoneoxycodone Pro-drug formation, Pro-drug formation, trypsin umols/h/trypsin umols/h/ half-life, h umol trypsin Com- half-life, h umoltrypsin Pro- Average ± Average ± pound Average ± Average ± drug sd sd109 sd sd KC-2 5.64 ± 0.26 37.4 ± 0.9 2 uM 116 ± 118 nd* *nd = notdetectable

The results in Table 4 indicate that trypsin can effect release ofoxycodone from a prodrug of the embodiments and that a trypsin inhibitorof the embodiments can attenuate trypsin-mediated release of oxycodone.

Example 20: Oral Administration of Compound KC-2 and Trypsin InhibitorCompound 109 to Rats

Saline solutions of Compound KC-2 (which can be prepared as described inExample 10) were dosed at 8.7 μmol/kg (6 mg/kg) with or without aco-dose of 55 μmol/kg (30 mg/kg) Compound 109 (Catalog No. 3081, TocrisBioscience, Ellisville, Mo., USA or Catalog No. WS38665, WaterstoneTechnology, Carmel, Ind., USA) as indicated in Table 5 via oral gavageinto jugular vein-cannulated male Sprague Dawley rats (4 per groups)that had been fasted for 16-18 hr prior to oral dosing. At specifiedtime points, blood samples were drawn, harvested for plasma viacentrifugation at 5,400 rpm at 4° C. for 5 min, and 100 microliters (μl)plasma transferred from each sample into a fresh tube containing 2 μl of50% formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored in −80° C. freezer until analysis byHPLC/MS.

Table 5 and FIG. 8 provide oxycodone exposure results for ratsadministered with Compound KC-2 in the absence or presence of trypsininhibitor. Results in Table 5 are reported as (a) maximum plasmaconcentration (Cmax) of oxycodone (OC) (average±standard deviation) and(b) time after administration of Compound KC-2 to reach maximumoxycodone concentration (Tmax) (average±standard deviation).

TABLE 5 Cmax and Tmax values of oxycodone in rat plasma KC-2 KC-2 Dose,Compound Compound Dose, μmol/ 109 Dose, 109 Dose, OC Cmax ± Tmax ± mg/kgkg mg/kg μmol/kg sd, ng/mL sd, hr 6 8.7 0 0 0.863 ± 0.69 3.00 ± 1.4 68.7 30 55 0.0468 ± 0.094 5.00 ± nc  Lower limit of quantitation was0.100 ng/mL; nc = not calculated

FIG. 8 compares mean plasma concentrations over time of oxycodonerelease following PO administration of Compound KC-2 with or without aco-dose of trypsin inhibitor.

The results in Table 5 and FIG. 8 indicate that Compound 109 attenuatesCompound KC-2's ability to release oxycodone, both by suppressing Cmaxand by delaying Tmax.

Example 21: Pharmacokinetics of Compound KC-2 Following POAdministration to Rats

Saline solutions of Compound KC-2 (which can be prepared as described inExample 10) were dosed as indicated in Table 6 via oral gavage intojugular vein-cannulated male Sprague Dawley rats (4 per group) that hadbeen fasted for 16-18 hr prior to oral dosing. At specified time points,blood samples were drawn, harvested for plasma via centrifugation at5,400 rpm at 4° C. for 5 min, and 100 microliters (μl) plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored in −80° C. freezer until analysis byHPLC/MS.

Table 6 and FIG. 9 provide oxycodone exposure results for ratsadministered with different doses of Compound KC-2. Results in Table 6are reported, for each group of rats, as (a) maximum plasmaconcentration (Cmax) of oxycodone (OC) (average+standard deviation), (b)time after administration of Compound KC-2 to reach maximum oxycodoneconcentration (Tmax) (average±standard deviation) and (c) area under thecurve (AUC) from 0 to 24 hr (average±standard deviation).

TABLE 6 Rat dosing PO with Compound KC-2 Dose, Dose OC Cmax ± Tmax ± AUC± sd, mg/kg μmol/kg sd, ng/mL sd, hr ng*hr/mL 1.3 1.9  0.144 ± 0.018 1.50 ± 0.58 0.445 ± 0.13 5 7.3 0.918 ± 0.30 2.75 ± 0.5 4.30 ± 1.1 6 8.70.863 ± 0.69 3.00 ± 1.4 4.29 ± 2.6 10 15  1.13 ± 0.75 3.75 ± 2.9 4.94 ±2.2 20 29 3.84 ± 1.1 3.75 ± 1.5 30.9 ± 6.3 42 61 6.00 ± 2.4 3.00 ± 1.439.6 ± 18  50 73 7.03 ± 2.3 3.75 ± 1.5 59.9 ± 14  Lower limit ofconcentration was 0.0500 ng/mL except 20 mg/kg dose was 0.0250 ng/mL

FIG. 9 compares mean plasma concentrations over time of oxycodonerelease following PO administration of increasing doses of CompoundKC-2.

The results in Figure Table 6 and 9 indicate that plasma concentrationsof oxycodone increase proportionally with Compound KC-2 dose.

Example 22: Oral Administration of Compound KC-2 Co-Dosed with TrypsinInhibitor Compound 109 to Rats

Saline solutions of Compound KC-2 were dosed at 7.3 μmol/kg (5 mg/kg)and 73 μmol/kg (50 mg/kg). The higher dose was co-dosed with increasingconcentrations of Compound 109 (Catalog No. 3081, Tocris Bioscience orCatalog No. WS38665, Waterstone Technology) as indicated in Table 7 viaoral gavage into jugular vein-cannulated male Sprague Dawley rats (4 pergroup) that had been fasted for 16-18 hr prior to oral dosing. Atspecified time points, blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 microliters(μl) plasma transferred from each sample into a fresh tube containing 2μl of 50% formic acid. The tubes were vortexed for 5-10 seconds,immediately placed in dry ice and then stored in −80° C. freezer untilanalysis by HPLC/MS.

Table 7 and FIG. 10 provide oxycodone exposure results for ratsadministered with different doses of Compound KC-2. Results in Table 7are reported, for each group of rats, as (a) maximum plasmaconcentration (Cmax) of oxycodone (OC) (average+standard deviation), (b)time after administration of Compound KC-2 to reach maximum oxycodoneconcentration (Tmax) (average±standard deviation) and (c) area under thecurve (AUC) from 0 to 24 hr (average±standard deviation).

TABLE 7 Rat dosing PO with Compound KC-2 in the absence or presence ofCompound 109 KC-2 KC-2 Compound Compound Dose, Dose, 109 Dose, 109 Dose,OC Cmax ± Tmax ± AUC ± sd, mg/kg μmol/kg mg/kg μmol/kg sd, ng/mL sd, hrng*hr/mL 5 7.3 0 0 0.918 ± 0.30 2.75 ± 0.5 4.30 ± 1.1 50 73 0 0 7.03 ±2.3 3.75 ± 1.5 59.9 ± 14  50 73 10 19 4.44 ± 1.5 6.50 ± 1.7 51.0 ± 16 50 73 20 37  2.25 ± 0.89 7.25 ± 1.5 29.2 ± 8.9 50 73 30 56  1.77 ± 0.576.50 ± 1.7 19.8 ± 7.6 50 73 40 74  1.64 ± 0.96 5.75 ± 1.5 16.5 ± 5.9Lower limit of quantitations were 0.0250 ng/ml

FIG. 10 compares mean plasma concentrations over time of oxycodonerelease following PO administration of Compound KC-2 with increasingamounts of co-dosed trypsin inhibitor Compound 109.

The results in Table 7 and FIG. 10 indicate Compound 109's ability toattenuate Compound KC-2's ability to release oxycodone in a dosedependent manner, both by suppressing Cmax and AUC and by delaying Tmax.

Example 23: In Vitro Human μ-Opioid Receptor Binding Assay

This example measures the affinity of compound KC-2 for the mu(p)-opioid receptor expressed in recombinant HEK-293 cells.

The general procedure follows the protocol described by Wang, J.-B.,Johnson, P. S., Perscio, A. M., Hawkins, A. L., Griffin, C. A. and Uhl,G. R. (1994). FEBS Lett., 338: 217-222. More specifically, the assaysincluded, as appropriate, oxycodone or Compound KC-2 (which can beprepared as described in Example 10) as well as recombinant HEK-293cells expressing the human μ-opioid receptor on their cell surfaces,reference compound [d-Ala²,N-Me-Phe⁴,Gly⁵⁻ol]-enkephalin (DAMGO),radioligand [³H]DAMGO (0.5 nM) and non-specific ligand naloxone (10 uM).The reaction mixtures were incubated at 22° C. for 2 hr. The sampleswere then submitted to scintillation counting.

In these assays, the specific binding of a test compound to thereceptors is defined as the difference between the total binding and thenon-specific binding determined in the presence of an excess ofunlabelled ligand. Results are expressed as a percent of control ofspecific binding and as a percent inhibition of control specific bindingobtained in the presence of test compounds. The IC₅₀ values(concentration of competing ligand required for 50% inhibition of[³H]DAMGO binding), and Hill coefficients (nH) were determined bynon-linear regression analysis of competition curves using Hill equationcurve fitting.

Table 8 shows the IC₅₀ values for oxycodone and Compound KC-2.

TABLE 8 IC₅₀ values Compound IC₅₀ Human μ-opioid receptor Oxycodone1.2E−08 Compound KC-2 2.2E−08

These data demonstrate that Compound KC-2 binds to the μ-opioid receptorwith an affinity about 2-fold less than that of oxycodone.

Example 24: In Vitro Human μ-Opioid Receptor Agonist Cellular FunctionalAssay

This Example measures the ability of certain compounds of the presentdisclosure to effect an agonist response when exposed to recombinanthuman μ-opioid receptor expressed in CHO cells.

The general procedure follows the protocol described by Wang, J.-B.,Johnson, P. S., Perscio, A. M., Hawkins, A. L., Griffin, C. A. and Uhl,G. R. (1994). FEBS Lett., 338: 217-222. More specifically, the assaysincluded each of the compounds indicated in Table 9 and recombinantChinese hamster ovary (CHO) cells expressing the human μ-opioid receptoron their cell surfaces. The control reaction included 1 M DAMGO. Thereaction mixtures were incubated at 37° C. for 10 min, and the reactionproduct was cyclic AMP (cAMP). The samples were submitted to homogeneoustime resolved fluorescence (HTRF®). EC₅₀ values (concentration producinga half-maximal specific response) were determined by non-linearregression fit using the Hillplot software.

Table 9 shows results from three separate experiments. EC₅₀ values areprovided for Compound KC-2, Compound KC-3, Compound KC-5, and CompoundKC-6 (each of which can be prepared as described in Examples, 10, 11, 13and 14, respectively) and compared to the EC₅₀ value for oxycodone,measured in the same respective experiments. Also shown are the EC₅₀values for Compound KC-4 (which can be prepared as described in Example12) and hydrocodone, measured in the same experiment. Table 9 alsoprovides the drug-to-prodrug (drug/prodrug) relative potency (i.e., EC₅₀at the human μ-opioid receptor) of oxycodone or hydrocodone to a prodrugof that respective drug.

TABLE 9 EC₅₀ values EC₅₀ Human μ- Drug/prodrug Experiment # Compoundopioid receptor relative potency 1 Oxycodone 1.2E−7 1 Compound KC-24.9E−7 4.1 2 Oxycodone 4.0E−8 2 Compound KC-3 1.6E−6 40 2 Compound KC-52.0E−6 50 3 Hydrocodone 8.8E−8 3 Compound KC-4 1.3E−6 15 3 Oxycodone7.8E−8 3 Compound KC-6 1.8E−6 23

The results of Table 9 show that prodrugs of the embodiments exhibit adrug/prodrug relative potency greater than 1; thus, prodrugs of theembodiments are less potent at the human t-opioid receptor than are therespective drugs they release.

Example 25: Pharmacokinetics Following IV Administration of CompoundKC-2 or Oxycodone to Rats: Plasma and Cerebrospinal Fluid Penetration

This Example compares the plasma and cerebrospinal fluid (CSF)concentrations of prodrug Compound KC-2 and oxycodone followingintravenous (IV) administration of the respective compounds to rats.Plasma/CSF partitioning coefficients are predictive of the ability of acompound to penetrate the blood-brain barrier.

Compound KC-2 (which can be prepared as described in Example 10), at adose of 10 mg/kg, or an equimolar dose of oxycodone each was dissolvedin saline and injected into the tail vein of 4 male Sprague Dawley rats.After 15 minutes, the rats were anesthetized by carbon dioxideasphyxiation and blood samples were drawn, harvested for plasma viacentrifugation at 5,400 rpm at 4° C. for 5 min, and 100 microliters (μl)plasma transferred from each sample into a fresh tube containing 2 μl of50% formic acid. The CSF fluid was collected using a 22×1 inch gaugeneedle connected to polyurethane catheter type MRE-040 tubing (BraintreeScientific, Inc., Braintree, Mass.). The needle was inserted just belowthe nuchal crest at the area of the foramen magnum; clear CSF fluid wascollected into the catheter and transferred into a collection tube. TheCSF samples were centrifuged at 5,400 rpm at 4° C. for 5 min, and 100 μlCSF fluid transferred from each sample into a fresh tube. The plasma andCSF samples were immediately placed in dry ice and then stored in a −80°C. freezer until analysis by high performance liquid chromatography/massspectrometry (HPLC/MS).

Results in Table 10 are reported, for each group of 4 rats as meanconcentrations of the indicated compounds in plasma or CSF. Table 10also provides the plasma-to-CSF (plasma/CSF) partitioning coefficient,i.e., the ratio of concentration in the plasma to concentration in theCSF of the indicated compounds.

TABLE 10 Mean plasma and CSF concentration values and partitioningcoefficients of Compound KC-2 and oxycodone Plasma/CSF Compound conc. inCompound conc. partitioning Compound Plasma, ng/mL in CSF, ng/mLcoefficient Compound KC-2 27,200 61.9 439 OC 3,257 863 3.8

The results in Table 10 indicate that the relative plasma/CSFpartitioning coefficient of Compound KC-2 to oxycodone is about 116(i.e., 439/3.8); that is, Compound KC-2 is about 116-fold less CSFpenetrant than oxycodone. In addition, as shown in Example 24, thedrug/prodrug relative potency of Compound KC-2 is about 4.1. Thus,Compound KC-2, when administered intravenously in equimolar amountswould be expected to be about 475-fold (i.e., 116×4.1) less effective atCNS mu-opioid receptors than oxycodone.

Example 26: Pharmacokinetics of Compound KC-3 Following POAdministration to Rats

This Example compares the pharmacokinetics of several concentrations ofCompound KC-3 administered orally (PO) to rats.

Saline solutions of Compound KC-3 (which can be prepared as described inExample 11) were dosed as indicated in Table 11 via oral gavage intojugular vein-cannulated male Sprague Dawley rats (4 per group, exceptdose 46 mg/kg KC-3 where 3 rats were used) that had been fasted for16-18 hr prior to oral dosing. At specified time points, blood sampleswere drawn, harvested for plasma via centrifugation at 5,400 rpm at 4°C. for 5 min, and 100 μl plasma transferred from each sample into afresh tube containing 2 μl of 50% formic acid. The tubes were vortexedfor 5-10 seconds, immediately placed in dry ice and then stored in −80°C. freezer until analysis by HPLC/MS.

Table 11 and FIG. 11 provide oxycodone exposure results for ratsadministered with different doses of Compound KC-3. Results in Table 11are reported, for each group of rats, as (a) maximum plasmaconcentration (Cmax) of oxycodone (OC) (average+standard deviation), (b)time after administration of Compound KC-3 to reach maximum oxycodoneconcentration (Tmax) (average±standard deviation) and (c) area under thecurve (AUC) from 0 to 24 hr for all doses except for the 1.4 mg/kg and22 mg/kg doses where the AUC values were calculated from 0 to 8 hr(average±standard deviation).

TABLE 11 Cmax, Tmax and AUC values of oxycodone in rat plasma Com- Dose,Dose OC Cmax ± sd, Tmax ± AUC ± sd pound mg/kg μmol/kg ng/mL sd, hr (ng× hr)/mL KC-3 1.4 1.9 0.0992 ± 0.0084 2.25 ± 0.5 0.376 ± 0.14 KC-3 11 151.34 ± 0.31 2.00 ± 0.0 8.96 ± 4.9 KC-3 22 30 2.54 ± 0.34 2.00 ± 0.0 12.6± 1.9 KC-3 46 63 5.19 ± 0.76 3.33 ± 1.5 40.5 ± 17  Lower limit ofquantitation was 0.05 ng/mL

FIG. 11 compares mean plasma concentrations over time of oxycodonerelease following PO administration of increasing doses of CompoundKC-3.

The results in Table 11 and FIG. 11 indicate that plasma concentrationsof oxycodone increase proportionally with Compound KC-3 dose.

Example 27: Pharmacokinetics of Compound KC-3 Following IVAdministration to Rats

This Example compares the plasma concentrations of prodrug and oxycodonein rats following intravenous (IV) administration of Compound KC-3.

Compound KC-3 (which can be prepared as described in Example 11) wasdissolved in saline and injected into the tail vein of 4 jugularvein-cannulated male Sprague Dawley rats at a dose of 2 mg/kg. Atspecified time points, blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 μl plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored in −80° C. freezer until analysis byhigh performance liquid chromatography/mass spectrometry (HPLC/MS).

Table 12 and FIG. 12 provide Compound KC-3 and oxycodone exposureresults for the group of rats administered Compound KC-3 intravenously.Results in Table 12 are reported as maximum plasma concentration (Cmax)of Compound KC-3 and oxycodone (OC), respectively (average±standarddeviation).

TABLE 12 Cmax values of Compound KC-3 and oxycodone in rat plasma KC-3KC-3 Dose, Dose, mg/kg μmol/kg KC-3 Cmax ± sd, ng/mL OC Cmax ± sd, ng/mL2 2.7 2620 ± 85 1.14 ± 0.48 Lower limit of quantitation was 0.05 ng/mL

Table 12 and FIG. 12 demonstrate that the plasma concentration ofoxycodone in rats administered Compound KC-3 intravenously is only 0.04%of the plasma concentration of Compound KC-3, indicating that IVadministration of Compound KC-3 does not lead to significant release ofoxycodone into plasma.

Example 28: Effect of Trypsin Inhibition on In Vitro Trypsin-MediatedTrypsin Release of Drug from Ketone-Modified Opioid Prodrugs

This Example demonstrates the ability of trypsin to cleave a prodrug ofthe embodiments and the effect of trypsin inhibitors on such cleavage.

Compound KC-3, Compound KC-4, Compound KC-5, or Compound KC-6 was eachincubated with trypsin from bovine pancreas (Catalog No. T8003, Type I,˜10,000 BAEE units/mg protein, Sigma-Aldrich). Specifically, thereactions included 0.761 mM of Compound KC-3.2HCl, Compound KC-5.2HCl,Compound KC-4.2HCl, or Compound KC-6.2HCl, 22.5 mM calcium chloride, 40to 172 mM Tris pH 8 and 0.25% DMSO with variable activities of trypsinas indicated in Table 13A. The reactions were conducted at 37° C. for 24hr. Samples were collected at specified time points, transferred into0.5% formic acid in acetonitrile to stop trypsin activity and stored atless than −70° C. until analysis by LC-MS/MS.

Compound KC-3 was also incubated in the presence of 2 micromolar (μM)trypsin inhibitor Compound 109. In that case, Compound KC-3 was added 5min after the other incubation components. Other reaction and sampletreatment conditions were as described above.

Tables 13A and 13B indicate the results of exposure of the testedcompounds to trypsin in the absence or presence of trypsin inhibitor.The results are expressed as half-life of prodrug when exposed totrypsin (i.e., Prodrug trypsin half-life) in hours and rate of oxycodoneor hydrocodone formation in umoles per hour per BAEE unit (umol/h/BAEEU) trypsin.

TABLE 13A In vitro trypsin conversion of prodrugs to oxycodone orhydrocodone Rate of Rate of hydrocodone BAEE Pro-drug oxycodoneformation, U trypsin formation, umol/h/ Pro- trypsin/ half-life, humol/h/BAEE U BAEE U drug mL Average ± sd Average ± sd Average ± sd KC-3241 8.92 ± 1.91 0.0684 ± 0.0009 na KC-5 241  1.2 ± 0.04 0.135 ± 0.005 naKC-4 241 6.35 ± 0.13 na 0.0911 ± 0.015  KC-4 4815 0.315 ± 0.004 na0.0137 ± 0.0014 KC-6 241 nc 0.0118 ± 0.0042 na KC-6 4815 nc 0.00571 ±0.0002  na nc = not calculable; na = not applicable

TABLE 13B Inhibition of in vitro trypsin conversion of Compound KC-3 tooxycodone by Compound 109 With trypsin inhibitor Pro-drug trypsin Rateof oxycodone formation, Compound half-life, h umol/h/BAEE U Prodrug 109Average ± sd Average ± sd KC-3 2 uM 43.338 ± 40.637 nc nc = notcalculable

The results in Table 13A indicate that trypsin can mediate release ofoxycodone or hydrocodone from a prodrug of the embodiments. The resultsin Table 13B indicate that a trypsin inhibitor of the embodiments canattenuate trypsin-mediated release of drug from a ketone-modified opioidprodrug of the embodiments.

Example 29: Oral Administration of Compound KC-3 and Trypsin InhibitorCompound 109 to Rats

This Example demonstrates the ability of a trypsin inhibitor of theembodiments to affect drug release into plasma from Compound KC-3administered orally.

Saline solutions of Compound KC-3 (which can be prepared as described inExample 11) were dosed at 6.8 μmol/kg (5 mg/kg) and 68 μmol/kg (50mg/kg) Compound KC-3 with or without a co-dose of increasingconcentrations of Compound 109 (Catalog No. 3081, Tocris Bioscience orCatalog No. WS38665, Waterstone Technology) as indicated in Table 14 viaoral gavage into jugular vein-cannulated male Sprague Dawley rats (4 pergroups) that had been fasted for 16-18 hr prior to oral dosing. Atspecified time points, blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 μl plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored in −80° C. freezer until analysis byHPLC/MS.

Table 14 and FIG. 13 provide oxycodone exposure results for ratsadministered with Compound KC-3 in the absence or presence of trypsininhibitor. Results in Table 14 are reported as (a) maximum plasmaconcentration (Cmax) of oxycodone (OC) (average±standard deviation), (b)time after administration of Compound KC-3 to reach maximum oxycodoneconcentration (Tmax) (average±standard deviation) and (c) area under thecurve (AUC) from 0 to 24 hr (average±standard deviation).

TABLE 14 Cmax, Tmax and AUC values of oxycodone in rat plasma KC-3 KC-3Compound Compound Dose, Dose, 109 Dose, 109 Dose, OC Cmax ± Tmax ± AUC ±sd mg/kg μmol/kg mg/kg μmol/kg sd, ng/mL sd, hr (ng × hr)/mL 5 6.8 0 00.611 ± 0.10  3.00 ± 1.4 3.95 ± 1.6 50 68 0 0 7.08 ± 2.6  3.00 ± 1.459.1 ± 23  50 68 10 18.5 1.26 ± 0.34 8.00 ± 0.0 12.3 ± 2.9 50 68 20 371.05 ± 0.61 3.75 ± 1.5 10.5 ± 5.4 50 68 30 55 0.49 ± 0.19 4.50 ± 2.62.82 ± 1.3 50 68 40 74 0.47 ± 0.36 4.63 ± 3.1 2.71 ± 3.7 Lower limit ofquantitation was 0.025 ng/mL

FIG. 13 compares mean plasma concentrations over time of oxycodonerelease following PO administration of Compound KC-3 with or without aco-dose of trypsin inhibitor.

The results in Table 14 and FIG. 13 indicate that Compound 109attenuates Compound KC-3's ability to release oxycodone, both bysuppressing Cmax and AUC and by delaying Tmax.

Example 30: Pharmacokinetics Following IV Administration of CompoundKC-3 or Oxycodone to Rats: Plasma and Cerebrospinal Fluid Penetration

This Example compares the plasma and cerebrospinal fluid (CSF)concentrations of prodrug Compound KC-3 and oxycodone followingintravenous (IV) administration of the respective compounds to rats.Plasma/CSF partitioning coefficients are predictive of the ability of acompound to penetrate the blood-brain barrier.

Compound KC-3 (which can be prepared as described in Example 11), at adose of 10 mg/kg, or an equimolar dose of oxycodone each was dissolvedin saline and injected into the tail vein of 4 male Sprague Dawley rats.After 2 minutes, the rats were anesthetized by carbon dioxideasphyxiation and blood samples were drawn, harvested for plasma viacentrifugation at 5,400 rpm at 4° C. for 5 min, and 100 μl plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The CSF fluid was collected using a 22×1 inch gauge needleconnected to polyurethane catheter type MRE-040 tubing (BraintreeScientific, Inc.). The needle was inserted just below the nuchal crestat the area of the foramen magnum; clear CSF fluid was collected intothe catheter and transferred into a collection tube. The CSF sampleswere centrifuged at 5,400 rpm at 4° C. for 5 min, and 100 μl CSF fluidtransferred from each sample into a fresh tube. The plasma and CSFsamples were immediately placed in dry ice and then stored in a −80° C.freezer until analysis by high performance liquid chromatography/massspectrometry (HPLC/MS). In order to study Compound KC-3 and oxycodoneplasma and CSF penetration over time, additional groups of 4 rats wereadministered compounds as described above and anesthetized at specifiedtime points. Plasma and CSF were collected and analyzed as describedabove. Results from these rats indicated that equilibrium was quicklyreached in the plasma and CSF compartments after dosing and that theextent of partitioning between CSF and plasma was consistent across timepoints. Thus, only the 2-minute time point data are reported in Table15.

Results in Table 15 are reported, for each group of 4 rats as meanconcentrations of the indicated compounds in plasma or CSF. Table 15also provides the plasma-to-CSF (plasma/CSF) partitioning coefficient,i.e., the ratio of concentration in the plasma to concentration in theCSF of the indicated compounds.

TABLE 15 Mean plasma and CSF concentration values and partitioningcoefficients of Compound KC-3 and oxycodone Compound Plasma/CSF conc. inCompound conc. in partitioning Compound Plasma, ng/mL CSF, ng/mLcoefficient Compound KC-3 59,225 34.1 1,737 OC 10,300 2158 4.8

The results in Table 15 indicate that the relative plasma/CSFpartitioning coefficient of Compound KC-3 to oxycodone is about 364(i.e., 1,737/4.8); that is, Compound KC-3 is about 364-fold less CSFpenetrant than oxycodone. In addition, as shown in Example 24, thedrug/prodrug relative potency of Compound KC-3 is about 40. Thus,Compound KC-3, when administered intravenously in equimolar amountswould be expected to be about 14,500-fold (i.e., 364×40) less effectiveat CNS mu-opioid receptors than oxycodone.

Example 31: In Vivo Tolerability of Compound KC-3 in Rats

This Example demonstrates that Compound KC-3 was tolerated whenadministered intravenously to rats.

Male naïve Sprague-Dawley rats, 4 per dose, were used in the study. Ratswere weighed, and then placed under a heat lamp for 15-20 minutes todilate the lateral tail veins. Dose volumes were based on the bodyweights (1 mL/kg); dosing of Compound KC-3 (which can be prepared asdescribed in Example 11) was as indicated in Table 16. Before dosing,rats were placed in Broome restrainers and the drug was introduced intoone of the tail veins using a syringe and needle. After dosing, thetimer was set and rats were observed for clinical signs. Blood sampleswere collected 5 minutes post-dose via the saphenous vein. The rats wereobserved up to 24 hours. Results are shown in Table 16.

TABLE 16 In vivo tolerability of Compound KC-3 in rats Dose, Dose,Number of Compound mg/kg μmol/kg Rats dosed Clinical observations KC-371 97 4 2 normal and 2 with ataxia which resolved by 2 minutes

The results in Table 16 indicate that rats tolerate a dose of 97 μmol/kgof Compound KC-3 and recover to normal activity within 2 minutes.

Example 32: In Vitro Stability of Oxycodone Prodrug Compound KC-3

This Example demonstrates the stability of Compound KC-3 to a variety ofreadily available household chemicals and enzyme preparations.

Compound KC-3 (which can be prepared as described in Example 11) wasexposed at room temperature (RT) or 80° C. for either 1 or 24 hours (hr)to the following household chemicals: vodka (40% alcohol), baking soda(saturated sodium bicarbonate solution, pH 9), WINDEX® with Ammonia-D(pH11) and vinegar (5% acetic acid). Compound KC-3 was also exposed tothe following enzyme-containing compositions at RT for 1 or 24 hr: GNC®Super Digestive (2 capsules of GNC Super Digestive Enzymes dissolved in5 mL of water), tenderizer (Adolf's meat tenderizer, primarily papain,dissolved in water to a concentration of 0.123 g/mL to approximate theconcentration of a marinade given on the bottle label), and subtilisn (8tablets of ULTRAZYME® contact lens cleaner (Advanced Medical Optics)dissolved in 4 mL water).

Samples were incubated as described. Aliquots were removed at 1 hr and24 hr and stabilized by adding each to a solution of 50% or 100% of 85%phosphoric acid solution to achieve a final pH of less than or equal topH 4. The stabilized aliquots were then diluted 4- to 6-fold with water,vortex-mixed and applied to HPLC.

FIG. 14 demonstrates the release of oxycodone when Compound KC-3 wasexposed to the various household chemicals and enzyme-containingcompositions described above. The percentage of Compound KC-3 remainingafter exposure is indicated by the solid black bars and percentageconversion of Compound KC-3 to oxycodone is indicated by the lightlyshaded bars with a black outline. These results indicate that exposureof Compound KC-3 to these various conditions leads to substantially lessthan 10% conversion to oxycodone.

Example 33: Pharmacokinetics of Compound KC-4 Following POAdministration to Rats

This Example demonstrates the release of hydrocodone into plasma whenCompound KC-4 is administered orally (PO) to rats.

Saline solutions of Compound KC-4 (which can be prepared as described inExample 12) were dosed as indicated in Table 17) via oral gavage intojugular vein-cannulated male Sprague Dawley rats (4 per group) that hadbeen fasted for 16-18 hr prior to oral dosing. At specified time points,blood samples were drawn, harvested for plasma via centrifugation at5,400 rpm at 4° C. for 5 min, and 100 μl plasma transferred from eachsample into a fresh tube containing 2 μl of 50% formic acid. The tubeswere vortexed for 5-10 seconds, immediately placed in dry ice and thenstored in −80° C. freezer until analysis by HPLC/MS.

Table 17 provides hydrocodone exposure results for rats administeredCompound KC-4 orally. Results in Table 17 are reported as (a) maximumplasma concentration (Cmax) of hydrocodone (OC) (average±standarddeviation), (b) time after administration of Compound KC-4 to reachmaximum hydrocodone concentration (Tmax) (average±standard deviation)and (c) area under the curve (AUC) from 0 to 24 hr (average±standarddeviation).

TABLE 17 Cmax, Tmax and AUC values of hydrocodone in rat plasma Dose,Dose mg/ μmol/ HC Cmax ± sd, Tmax ± AUC ± sd Compound kg kg ng/mL sd, hr(ng × hr)/mL KC-4 6 8.4 0.0667 ± 0.019 4.5 ± 2.6 0.315 ± 0.063 Lowerlimit of quantitation was 0.025 ng/mL

The results in Table 17 indicate that oral administration of CompoundKC-4 leads to release of hydrocodone by a hydrocodone prodrug of theembodiments.

Example 34: Pharmacokinetics of Compound KC-4 Following IVAdministration to Rats

This Example compares the plasma concentrations of prodrug andhydrocodone in rats following intravenous (IV) administration ofCompound KC-4.

Compound KC-4 (which can be prepared as described in Example 14) wasdissolved in saline and injected into the tail vein of 4 jugularvein-cannulated male Sprague Dawley rats at a dose of 2 mg/kg. Atspecified time points, blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 μl plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored in −80° C. freezer until analysis byhigh performance liquid chromatography/mass spectrometry (HPLC/MS).

Table 18 and FIG. 15 provide Compound KC-4 and hydrocodone exposureresults for rats administered Compound KC-4 intravenously. Results inTable 18 are reported as maximum plasma concentration (Cmax) of CompoundKC-4 and hydrocodone (HC), respectively, (average±standard deviation).

TABLE 18 Cmax values of Compound KC-4 and hydrocodone in rat plasma KC-4KC-4 Dose, Dose, KC-4 Cmax ± sd, mg/kg μmol/kg ng/mL* HC Cmax ± sd,ng/mL{circumflex over ( )} 2 2.8 3960 ± 570 0.224 ± 0.020 *Lower limitof quantitation was 0.05 ng/mL {circumflex over ( )}Lower limit ofquantitation was 0.025 ng/mL

Table 18 and FIG. 15 demonstrate that the plasma concentration ofhydrocodone in rats administered Compound KC-4 intravenously is only0.006% of the plasma concentration of Compound KC-4, indicating that IVadministration of Compound KC-4 does not lead to significant release ofhydrocodone into plasma.

Example 35: Oral Administration of Compound KC-4 and Trypsin InhibitorCompound 109 to Rats

This Example demonstrates the ability of a trypsin inhibitor of theembodiments to affect drug release into plasma from Compound KC-4administered orally.

Saline solutions of Compound KC-4 (which can be prepared as described inExample 12) were dosed at 8.4 μmol/kg (6 mg/kg) with or without aco-dose of 55 μmol/kg (30 mg/kg) Compound 109 (Catalog No. 3081, TocrisBioscience or Catalog No. WS38665, Waterstone Technology) as indicatedin Table 19 via oral gavage into jugular vein-cannulated male SpragueDawley rats (4 per groups) that had been fasted for 16-18 hr prior tooral dosing. At specified time points, blood samples were drawn,harvested for plasma via centrifugation at 5,400 rpm at 4° C. for 5 min,and 100 μl plasma transferred from each sample into a fresh tubecontaining 2 μl of 50% formic acid. The tubes were vortexed for 5-10seconds, immediately placed in dry ice and then stored in −80° C.freezer until analysis by HPLC/MS.

Table 19 and FIG. 16 provide hydrocodone exposure results for ratsadministered with Compound KC-4 in the absence or presence of trypsininhibitor. Results in Table 19 are reported as (a) maximum plasmaconcentration (Cmax) of hydrocodone (HC) (average±standard deviation),(b) time after administration of Compound KC-4 to reach maximumhydrocodone concentration (Tmax) (average±standard deviation) and (c)area under the curve from 0 to 24 hr (average±standard deviation).

TABLE 19 Cmax, Tmax and AUC values of hydrocodone in rat plasma KC-4KC-4 Compound Compound Dose, Dose, 109 Dose, 109 Dose, HC Cmax ± Tmax ±AUC ± sd mg/kg μmol/kg mg/kg μmol/kg sd, ng/mL sd, hr (ng × hr)/mL 6 8.40 0 0.0667 ± 0.019 4.5 ± 2.6 0.315 ± 0.063 6 8.4 30 55 0.0064 ± 0.0138.0 ± 0.0 0.016 ± 0.032 Lower limit of quantitation was 0.025 ng/mL

FIG. 16 compares mean plasma concentrations over time of hydrocodonerelease following PO administration of Compound KC-4 with or without aco-dose of trypsin inhibitor.

The results in Table 19 and FIG. 16 indicate that Compound 109attenuates Compound KC-4's ability to release hydrocodone, both bysuppressing Cmax and AUC and by delaying Tmax.

Example 36: Pharmacokinetics of Compound KC-5 Following POAdministration to Rats

This Example demonstrates the release of oxycodone into plasma whenCompound KC-5 is administered orally (PO) to rats.

Saline solutions of Compound KC-5 (which can be prepared as described inExample 13) were dosed as indicated in Table 20 via oral gavage intojugular vein-cannulated male Sprague Dawley rats (4 per group) that hadbeen fasted for 16-18 hr prior to oral dosing. At specified time points,blood samples were drawn, harvested for plasma via centrifugation at5,400 rpm at 4° C. for 5 min, and 100 μl plasma transferred from eachsample into a fresh tube containing 2 μl of 50% formic acid. The tubeswere vortexed for 5-10 seconds, immediately placed in dry ice and thenstored in −80° C. freezer until analysis by HPLC/MS.

Table 20 and FIG. 17 provide oxycodone exposure results for ratsadministered Compound KC-5 orally. Results in Table 20 are reported as(a) maximum plasma concentration (Cmax) of oxycodone (OC)(average±standard deviation), (b) time after administration of CompoundKC-5 to reach maximum oxycodone concentration (Tmax) (average±standarddeviation) and (c) area under the curve (AUC) (ng×hr)/mL from 0 to 8 hr(average±standard deviation).

TABLE 20 Cmax, Tmax and AUC values of oxycodone (OC) in rat plasma Dose,Dose OC Cmax ± Tmax ± AUC ± sd Compound mg/kg μmol/kg sd, ng/mL sd, hr(ng × hr)/mL KC-5 24 30.5 2.06 ± 0.45 2.0 ± 0.0 9.61 ± 1.4 Lower limitof quantitation was 0.025 ng/mL

FIG. 17 demonstrates mean plasma concentrations over time of oxycodonerelease following PO administration of Compound KC-5.

The results in Table 20 and FIG. 17 indicate that oral administration ofCompound KC-5 yields oxycodone plasma concentrations that exhibit asuppressed Cmax and AUC and delayed Tmax compared to administration ofoxycodone (see Example 15).

Example 37 Pharmacokinetics of Compound KC-5 Following IV Administrationto Rats

This Example compares the plasma concentrations of prodrug and oxycodonein rats following intravenous (IV) administration of Compound KC-5.

Compound KC-5 (which can be prepared as described in Example 13) wasdissolved in saline and injected into the tail vein of 4 jugularvein-cannulated male Sprague Dawley rats at a dose of 2 mg/kg. Atspecified time points, blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 μl plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored in −80° C. freezer until analysis byhigh performance liquid chromatography/mass spectrometry (HPLC/MS).

Table 21 and FIG. 18 provide Compound KC-5 and oxycodone exposureresults for rats administered Compound KC-5 intravenously. Results inTable 21 are reported as maximum plasma concentration (Cmax) of CompoundKC-5 and oxycodone (OC), respectively (average±standard deviation).

TABLE 21 Cmax values of Compound KC-5 and oxycodone in rat plasma KC-5KC-5 Dose, Dose, mg/kg μmol/kg KC-5 Cmax ± sd, ng/mL* OC Cmax ± sd,ng/mL{circumflex over ( )} 2 2.5 3140 ± 270 0.878 ± 0.78 *Lower limit ofquantitation was 0.100 ng/mL {circumflex over ( )}Lower limit ofquantitation was 0.0125 ng/mL

Table 21 and FIG. 18 demonstrate that the plasma concentration ofoxycodone in rats administered Compound KC-5 IV is only 0.028% of theplasma concentration of Compound KC-5, indicating that IV administrationof Compound KC-5 does not lead to significant release of oxycodone intoplasma.

Example 38: Pharmacokinetics Following IV Administration of CompoundKC-5 or Oxycodone to Rats: Plasma and Cerebrospinal Fluid Penetration

This Example compares the plasma and cerebrospinal fluid (CSF)concentrations of prodrug Compound KC-5 and oxycodone followingintravenous (IV) administration of the respective compounds to rats.Plasma/CSF partitioning coefficients are predictive of the ability of acompound to penetrate the blood-brain barrier.

Compound KC-5 (which can be prepared as described in Example 13), at adose of 10 mg/kg, or an equimolar dose of oxycodone each was dissolvedin saline and injected into the tail vein of 4 male Sprague Dawley rats.After 2 minutes, the rats were anesthetized by carbon dioxideasphyxiation and blood samples were drawn, harvested for plasma viacentrifugation at 5,400 rpm at 4° C. for 5 min, and 100 μl plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The CSF fluid was collected using a 22×1 inch gauge needleconnected to polyurethane catheter type MRE-040 tubing (BraintreeScientific, Inc.). The needle was inserted just below the nuchal crestat the area of the foramen magnum and clear CSF fluid was collected intothe catheter and transferred into a collection tube. The CSF sampleswere centrifuged at 5,400 rpm at 4° C. for 5 min, and 100 μl CSF fluidtransferred from each sample into a fresh tube. The plasma and CSFsamples were immediately placed in dry ice and then stored in a −80° C.freezer until analysis by high performance liquid chromatography/massspectrometry (HPLC/MS). In order to study Compound KC-5 and oxycodoneplasma and CSF penetration over time, additional groups of 4 rats wereadministered compounds as described above and anesthetized at specifiedtime points. Plasma and CSF were collected and analyzed as describedabove. Results from these rats indicated that equilibrium was quicklyreached in the plasma and CSF compartments after dosing and that theextent of partitioning between CSF and plasma was consistent across timepoints. Thus, only the 2-minute time point data are reported in Table22.

Results in Table 22 are reported, for each group of 4 rats as meanconcentrations of the indicated compounds in plasma or CSF. Table 22also provides the plasma-to-CSF (plasma/CSF) partitioning coefficient,i.e., the ratio of concentration in the plasma to concentration in theCSF of the indicated compounds.

TABLE 22 Mean plasma and CSF concentration values and partitioningcoefficients of Compound KC-5 and oxycodone Plasma/CSF Compound conc.Compound conc. in partitioning Compound in Plasma, ng/mL CSF, ng/mLcoefficient Compound KC-5 54,900 36.4 1,508 OC 10,300 2,158 4.8

The results in Table 22 indicate that the relative plasma/CSFpartitioning coefficient of Compound KC-5 to oxycodone is about 316(i.e., 1,508/4.8); that is, Compound KC-5 is about 316-fold less CSFpenetrant than oxycodone. In addition, as shown in Example 24, thedrug/prodrug relative potency of Compound KC-5 is about 50. Thus,Compound KC-5, when administered intravenously in equimolar amountswould be expected to be about 15,800-fold (i.e., 316×50) less effectiveat CNS mu-opioid receptors than oxycodone.

Example 39: Pharmacokinetics of Compound KC-6 Following POAdministration to Rats

This Example demonstrates the release of oxycodone into plasma whenCompound KC-6 is administered orally (PO) to rats.

Saline solutions of Compound KC-6 (which can be prepared as described inExample 14) were dosed as indicated in Table 23 via oral gavage intojugular vein-cannulated male Sprague Dawley rats (4 per group) that hadbeen fasted for 16-18 hr prior to oral dosing. At specified time points,blood samples were drawn, harvested for plasma via centrifugation at5,400 rpm at 4° C. for 5 min, and 100 μl plasma transferred from eachsample into a fresh tube containing 2 μl of 50% formic acid. The tubeswere vortexed for 5-10 seconds, immediately placed in dry ice and thenstored in −80° C. freezer until analysis by HPLC/MS.

Table 23 and FIG. 19 provide oxycodone exposure results for ratsadministered Compound KC-6 orally. Results in Table 23 are reported as(a) maximum plasma concentration (Cmax) of oxycodone (OC)(average±standard deviation), (b) time after administration of CompoundKC-6 to reach maximum oxycodone concentration (Tmax) (average±standarddeviation) and (c) area under the curve (AUC) (ng×hr)/mL from 0 to 8 hr(average±standard deviation).

TABLE 23 Cmax, Tmax and AUC values of oxycodone in rat plasma Com- Dose,Dose OC Cmax ± AUC ± sd pound mg/kg μmol/kg sd, ng/mL Tmax ± sd, hr (ng× hr)/mL KC-6 24 30 2.72 ± 0.18 4.25 ± 1.5 15.1 ± 0.75 Lower limit ofquantitation was 0.025 ng/mL

FIG. 19 demonstrates mean plasma concentrations over time of oxycodonerelease following PO administration of Compound KC-6.

The results in Table 23 and FIG. 19 indicate that oral administration ofCompound KC-6 yields oxycodone plasma concentrations that exhibit asuppressed Cmax and AUC and delayed Tmax compared to administration ofoxycodone (see Example 15).

Example 40: Pharmacokinetics of Compound KC-6 Following IVAdministration to Rats

This Example compares the plasma concentrations of prodrug and oxycodonein rats following intravenous (IV) administration of Compound KC-6.

Compound KC-6 (which can be prepared as described in Example 14) wasdissolved in saline and injected into the tail vein of 4 jugularvein-cannulated male Sprague Dawley rats at a dose of 2 mg/kg. Atspecified time points, blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 μl plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice and then stored in −80° C. freezer until analysis byhigh performance liquid chromatography/mass spectrometry (HPLC/MS).

Table 24 and FIG. 20 provide Compound KC-6 and oxycodone exposureresults for rats administered Compound KC-6 intravenously. Results inTable 24 are reported as maximum plasma concentration (Cmax) of CompoundKC-6 and oxycodone (OC), respectively, (average±standard deviation).

TABLE 24 Cmax values of Compound KC-6 and oxycodone in rat plasma KC-6KC-6 Dose, Dose, KC-6 Cmax ± sd, mg/kg μmol/kg ng/mL OC Cmax ± sd, ng/mL2 2.5 6360 ± 2300* 0.960 ± 0.22{circumflex over ( )} *Lower limit ofquantitation was 0.05 ng/mL {circumflex over ( )}Lower limit ofquantitation was 0.1 ng/mL

Table 24 and FIG. 20 demonstrate that the plasma concentration ofoxycodone in rats administered Compound KC-6 intravenously is only0.015% of the plasma concentration of Compound KC-6, indicating that IVadministration of Compound KC-6 does not lead to significant release ofoxycodone into plasma.

Example 41: Pharmacokinetics Following IV Administration of CompoundKC-6 to Rats: Plasma and Cerebrospinal Fluid Penetration

This Example compares the plasma and cerebrospinal fluid (CSF)concentrations of prodrug Compound KC-6 and oxycodone followingintravenous (IV) administration of the respective compounds to rats.Plasma/CSF partitioning coefficients are predictive of the ability of acompound to penetrate the blood-brain barrier.

Compound KC-6 (which can be prepared as described in Example 14), at adose of 7.5 mg/kg, and oxycodone at a dose of 7.5 mg/kg, each wasdissolved in saline and injected into the tail vein of 4 male SpragueDawley rats. After 2 minutes, the rats were anesthetized by carbondioxide asphyxiation and blood samples were drawn, harvested for plasmavia centrifugation at 5,400 rpm at 4° C. for 5 min, and 100 μl plasmatransferred from each sample into a fresh tube containing 2 μl of 50%formic acid. The CSF fluid was collected using a 22×1 inch gauge needleconnected to polyurethane catheter type MRE-040 tubing (BraintreeScientific, Inc.). The needle was inserted just below the nuchal crestat the area of the foramen magnum and clear CSF fluid was collected intothe catheter and transferred into a collection tube. The CSF sampleswere centrifuged at 5,400 rpm at 4° C. for 5 min, and 100 μl CSF fluidtransferred from each sample into a fresh tube. The plasma and CSFsamples were immediately placed in dry ice and then stored in a −80° C.freezer until analysis by high performance liquid chromatography/massspectrometry (HPLC/MS). In order to study Compound KC-6 and oxycodoneplasma and CSF penetration over time, additional groups of 4 rats wereadministered compounds as described above and anesthetized at specifiedtime points. Plasma and CSF were collected and analyzed as describedabove. Results from these rats indicated that equilibrium was quicklyreached in the plasma and CSF compartments after dosing and that theextent of partitioning between CSF and plasma was consistent across timepoints. Thus, only the 2-minute time point data are reported in Table25.

Results in Table 25 are reported, for each group of 4 rats as meanconcentrations of the indicated compounds in plasma or CSF. Table 25also provides the plasma-to-CSF (plasma/CSF) partitioning coefficient,i.e., the ratio of concentration in the plasma to concentration in theCSF of the indicated compounds.

TABLE 25 Mean plasma and CSF concentration values and partitioningcoefficients of Compound KC-6 and oxycodone Plasma/CSF Compound conc.Compound conc. in partitioning Compound in Plasma, ng/mL CSF, ng/mLcoefficient Compound KC-6 60,400 74.1 815 OC 10,300 2,158 4.8

The results in Table 25 indicate that the relative plasma/CSFpartitioning coefficient of Compound KC-6 to oxycodone is about 171(i.e., 815/4.8); that is, Compound KC-6 is about 171-fold less CSFpenetrant than oxycodone. In addition, as shown in Example 24, thedrug/prodrug relative potency of Compound KC-6 is about 23. Thus,Compound KC-6, when administered intravenously in equimolar amountswould be expected to be about 3,940-fold (i.e., 171×23) less effectiveat CNS mu-opioid receptors than oxycodone.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-103. (canceled)
 104. A composition comprising: (i) a trypsininhibitor; and (ii) a compound of formula KC-(IIIa):

wherein: X represents a residue of a ketone-containing opioid, whereinthe hydrogen atom of the corresponding enolic group of the ketone isreplaced by a covalent bond to —C(O)—NR⁵—(C(R¹)(R²))_(n)—NR³R⁴; R⁵ isselected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl; each R¹ is independently selectedfrom hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, acyl,and aminoacyl; each R² is independently selected from hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, acyl, and aminoacyl; or R¹and R² together with the carbon to which they are attached form acycloalkyl, substituted cycloalkyl, aryl, or substituted aryl group, ortwo R¹ and R² groups on adjacent carbon atoms, together with the carbonatoms to which they are attached, form a cycloalkyl, substitutedcycloalkyl, aryl, or substituted aryl group; n is an integer from 2 to4; R³ is hydrogen; R⁴ is

each R⁶ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring; each W isindependently —NR⁸—, —O— or —S—; each R⁸ is independently selected fromhydrogen, alkyl, substituted alkyl, aryl and substituted aryl, oroptionally, each R⁶ and R⁸ independently together with the atoms towhich they are bonded form a cycloheteroalkyl or substitutedcycloheteroalkyl ring; p is an integer from one to 100; and R⁷ isselected from hydrogen, alkyl, substituted alkyl, acyl, substitutedacyl, alkoxycarbonyl, substituted alkoxycarbonyl, aryl, substitutedaryl, arylalkyl, and substituted arylalkyl; or a salt, hydrate orsolvate thereof; or. (i) a trypsin inhibitor; and (ii) a compound offormula KC-(IIIb):

wherein: X represents a residue of a ketone-containing opioid, whereinthe hydrogen atom of the corresponding enolic group of the ketone isreplaced by a covalent bond to —C(O)—NR⁵—(C(R¹)(R²))_(n)—NR³R⁴; R⁵ isselected from alkyl, substituted alkyl, arylalkyl, substitutedarylalkyl, aryl and substituted aryl; each R¹ is independently selectedfrom hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, acyl,and aminoacyl; each R² is independently selected from hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, acyl, and aminoacyl; or R¹and R² together with the carbon to which they are attached form acycloalkyl or substituted cycloalkyl group, or two R¹ or R² groups onadjacent carbon atoms, together with the carbon atoms to which they areattached, form a cycloalkyl or substituted cycloalkyl group; n is aninteger from 2 to 4; R³ is hydrogen; R⁴ is

each R⁶ is independently selected from hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heteroalkyl, substituted heteroalkyl, heteroaryl, substitutedheteroaryl, heteroarylalkyl, and substituted heteroarylalkyl, oroptionally, R⁶ and R⁷ together with the atoms to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring; each W isindependently —NR⁸—, —O— or —S—; each R⁸ is independently selected fromhydrogen, alkyl, substituted alkyl, aryl and substituted aryl, oroptionally, each R⁶ and R⁸ independently together with the atoms towhich they are bonded form a cycloheteroalkyl or substitutedcycloheteroalkyl ring; p is an integer from one to 100; and R⁷ isselected from hydrogen, alkyl, substituted alkyl, acyl, substitutedacyl, alkoxycarbonyl, substituted alkoxycarbonyl, aryl, substitutedaryl, arylalkyl, and substituted arylalkyl; or a salt, hydrate orsolvate thereof.
 105. The composition of claim 104, wherein theketone-containing opioid is selected from acetylmorphine, hydrocodone,hydromorphone, ketobemidone, methadone, naloxone, N-methylnaloxone,naltrexone, N-methylnaltrexone, oxycodone, oxymorphone, andpentamorphine.
 106. The composition of claim 104, wherein theketone-containing opioid is hydrocodone or oxycodone.
 107. Thecomposition of claim 104, wherein R⁵ is (1-4C)alkyl.
 108. Thecomposition of claim 107, wherein R⁵ is methyl or ethyl.
 109. Thecomposition of claim 104, wherein R¹ and R² are hydrogen.
 110. Thecomposition of claim 104, wherein R¹ and R² which are on the same carbonare methyl.
 111. The composition of claim 104, wherein one of R¹ and R²is aminoacyl.
 112. The composition of claim 104, wherein one of R¹ andR² is —C(O)NR^(10a)R^(10b), wherein each R^(10a) and R^(10b) isindependently selected from hydrogen, alkyl, substituted alkyl, andacyl.
 113. The composition of claim 104, wherein one of R¹ and R² is—C(O)NR^(10a)R^(10b), wherein R^(10a) is an alkyl and R^(10b) issubstituted alkyl.
 114. The composition of claim 104, wherein n is 2 or3.
 115. The composition of claim 104, wherein R⁴ is a residue of anL-amino acid selected from alanine, arginine, asparagine, aspartic acid,cysteine, glycine, glutamine, glutamic acid, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine and valine, or a residue of an N-acyl derivative ofany of said amino acids; or a residue of a peptide composed of at leasttwo L-amino acid residues selected independently from alanine, arginine,asparagine, aspartic acid, cysteine, glycine, glutamine, glutamic acid,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine and valine or a residueof an N-acyl derivative thereof.
 116. The composition of claim 115,wherein the N-acyl derivative is an acetyl, benzoyl, malonyl, piperonylor succinyl derivative.
 117. The composition of claim 104, wherein R⁴ isa residue of L-arginine or L-lysine, or a residue of an N-acylderivative of L-arginine or L-lysine.
 118. The composition of claim 104,wherein R⁶ is a side chain of an amino acid.
 119. The composition ofclaim 104, wherein R⁶ is —H, —CH₂CH₂CH₂NH(C═NH)NH₂ or —CH₂CH₂CH₂CH₂NH₂.120. The composition of claim 104, wherein R⁷ is selected from hydrogen,acetyl, benzoyl, malonyl, piperonyl and succinyl.
 121. The compositionof claim 104, wherein the compound is selected from the followingformulae:


122. The composition of claim 104, wherein the trypsin inhibitor is anarginine mimic or a lysine mimic.
 123. A method of treating orpreventing pain comprising administering an effective amount of acomposition of claim 104 to a patient in need thereof.